LOCUS       sequence-491690-       10337 bp    DNA     circular SYN 15-DEC-2025
DEFINITION  Mammalian expression of a human codon optimized wildtype Dn29 
            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-Dn29-dCas9-P2A-GFP
FEATURES             Location/Qualifiers
     source          1..10337
                     /mol_type="other DNA"
                     /organism="synthetic DNA construct"
     rep_origin      780..1368
                     /direction=RIGHT
                     /label=ori
                     /note="high-copy-number ColE1/pMB1/pBR322/pUC origin of 
                     replication"
     primer_bind     1269..1288
                     /label=pBR322ori-F
                     /note="pBR322 origin, forward primer"
     misc_RNA        complement(1501..1576)
                     /label=gRNA scaffold
                     /note="guide RNA scaffold for the Streptococcus pyogenes 
                     CRISPR/Cas9 system"
     promoter        complement(1597..1845)
                     /label=U6 promoter
                     /note="RNA polymerase III promoter for human U6 snRNA 
                     (Domitrovich & Kunkel, 2003)"
     primer_bind     complement(1655..1674)
                     /label=LKO.1 5'
                     /note="Human U6 promoter, forward primer"
     primer_bind     complement(1825..1845)
                     /label=hU6-F
                     /note="Human U6 promoter, forward primer"
     primer_bind     1896..1913
                     /label=L4440
                     /note="L4440 vector, forward primer"
     promoter        1958..3136
                     /label=EF-1-alpha promoter
                     /note="strong constitutive promoter for human elongation 
                     factor EF-1-alpha"
     intron          2189..3127
                     /label=EF-1-alpha intron A
                     /note="intron upstream of the start codon of human 
                     EF-1-alpha"
     primer_bind     3084..3104
                     /label=EF1a-F
                     /note="Human elongation factor-1a promoter, forward primer"
     regulatory      3143..3152
                     /label=Kozak sequence
                     /note="vertebrate consensus sequence for strong initiation 
                     of translation (Kozak, 1987)"
                     /regulatory_class="other"
     CDS             4709..4729
                     /codon_start=1
                     /product="nuclear localization signal of SV40 (simian virus
                     40) large T antigen"
                     /label=SV40 NLS
                     /translation="PKKKRKV"
     CDS             4856..4876
                     /codon_start=1
                     /product="nuclear localization signal of SV40 (simian virus
                     40) large T antigen"
                     /label=SV40 NLS
                     /translation="PKKKRKV"
     CDS             join(4886..4909,4910..4912,4913..7399,7400..7402,
                     7403..8986)
                     /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             4886..8986
                     /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             8990..9010
                     /codon_start=1
                     /product="nuclear localization signal of SV40 (simian virus
                     40) large T antigen"
                     /label=SV40 NLS
                     /translation="PKKKRKV"
     CDS             9026..9079
                     /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(9080..9082,9083..9796)
                     /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(9080..9082,9083..9793)
                     /codon_start=1
                     /product="enhanced GFP"
                     /label=EGFP
                     /translation="VSKGEELFTGVVPILVELDGDVNGHKFSVSGEGEGDATYGKLTLK
                     FICTTGKLPVPWPTLVTTLTYGVQCFSRYPDHMKQHDFFKSAMPEGYVQERTIFFKDDG
                     NYKTRAEVKFEGDTLVNRIELKGIDFKEDGNILGHKLEYNYNSHNVYIMADKQKNGIKV
                     NFKIRHNIEDGSVQLADHYQQNTPIGDGPVLLPDNHYLSTQSALSKDPNEKRDHMVLLE
                     FVTAAGITLGMDELYK"
     primer_bind     complement(9383..9402)
                     /label=EXFP-R
                     /note="For distinguishing EGFP variants, reverse primer"
     primer_bind     9730..9751
                     /label=EGFP-C
                     /note="EGFP, forward primer"
     polyA_signal    9828..9949
                     /label=SV40 poly(A) signal
                     /note="SV40 polyadenylation signal"
     primer_bind     complement(9865..9884)
                     /label=SV40pA-R
                     /note="SV40 polyA, reverse primer"
     primer_bind     9919..9938
                     /label=EBV-rev
                     /note="SV40 polyA terminator, reverse primer"
     promoter        9981..10085
                     /gene="bla"
                     /label=AmpR promoter
     CDS             join(10086..10154,10155..10337,1..609)
                     /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(10304..10323)
                     /label=Amp-R
                     /note="Ampicillin resistance gene, reverse primer"
ORIGIN
        1 gggcaagagc aactcggtcg ccgcatacac tattctcaga atgacttggt tgagtactca
       61 ccagtcacag aaaagcatct tacggatggc atgacagtaa gagaattatg cagtgctgcc
      121 ataaccatga gtgataacac tgcggccaac ttacttctga caacgatcgg aggaccgaag
      181 gagctaaccg cttttttgca caacatgggg gatcatgtaa ctcgccttga tcgttgggaa
      241 ccggagctga atgaagccat accaaacgac gagcgtgaca ccacgatgcc tgtagcaatg
      301 gcaacaacgt tgcgcaaact attaactggc gaactactta ctctagcttc ccggcaacaa
      361 ttaatagact ggatggaggc ggataaagtt gcaggaccac ttctgcgctc ggcccttccg
      421 gctggctggt ttattgctga taaatctgga gccggtgagc gtgggtctcg cggtatcatt
      481 gcagcactgg ggccagatgg taagccctcc cgtatcgtag ttatctacac gacggggagt
      541 caggcaacta tggatgaacg aaatagacag atcgctgaga taggtgcctc actgattaag
      601 cattggtaac tgtcagacca agtttactca tatatacttt agattgattt aaaacttcat
      661 ttttaattta aaaggatcta ggtgaagatc ctttttgata atctcatgac caaaatccct
      721 taacgtgagt tttcgttcca ctgagcgtca gaccccgtag aaaagatcaa aggatcttct
      781 tgagatcctt tttttctgcg cgtaatctgc tgcttgcaaa caaaaaaacc accgctacca
      841 gcggtggttt gtttgccgga tcaagagcta ccaactcttt ttccgaaggt aactggcttc
      901 agcagagcgc agataccaaa tactgttctt ctagtgtagc cgtagttagg ccaccacttc
      961 aagaactctg tagcaccgcc tacatacctc gctctgctaa tcctgttacc agtggctgct
     1021 gccagtggcg ataagtcgtg tcttaccggg ttggactcaa gacgatagtt accggataag
     1081 gcgcagcggt cgggctgaac ggggggttcg tgcacacagc ccagcttgga gcgaacgacc
     1141 tacaccgaac tgagatacct acagcgtgag ctatgagaaa gcgccacgct tcccgaaggg
     1201 agaaaggcgg acaggtatcc ggtaagcggc agggtcggaa caggagagcg cacgagggag
     1261 cttccagggg gaaacgcctg gtatctttat agtcctgtcg ggtttcgcca cctctgactt
     1321 gagcgtcgat ttttgtgatg ctcgtcaggg gggcggagcc tatggaaaaa cgccagcaac
     1381 gcggcctttt tacggttcct ggccttttgc tggccttttg ctcacatgtt ctttcctgcg
     1441 ttatcccctg attctgtgga taaccgtatt accgcctttg cgcatcagaa ttcaaaaaaa
     1501 gcaccgactc ggtgccactt tttcaagttg ataacggact agccttattt taacttgcta
     1561 tttctagctc taaaacacaa cagcagaata cacactggtg tttcgtcctt tccacaagat
     1621 atataaagcc aagaaatcga aatactttca agttacggta agcatatgat agtccatttt
     1681 aaaacataat tttaaaactg caaactaccc aagaaattat tactttctac gtcacgtatt
     1741 ttgtactaat atctttgtgt ttacagtcaa attaattcca attatctctc taacagcctt
     1801 gtatcgtata tgcaaatatg aaggaatcat gggaaatagg ccctcgctag ctgaagtgag
     1861 ctgataccgc tcgccgcagc cgaacgaccg agcgcagcga gtcagtgagc gaggaaggga
     1921 agagcggacc ttctaggtct tgaaaggagt gggaattggc tccggtgccc gtcagtgggc
     1981 agagcgcaca tcgcccacag tccccgagaa gttgggggga ggggtcggca attgaaccgg
     2041 tgcctagaga aggtggcgcg gggtaaactg ggaaagtgat gtcgtgtact ggctccgcct
     2101 ttttcccgag ggtgggggag aaccgtatat aagtgcagta gtcgccgtga acgttctttt
     2161 tcgcaacggg tttgccgcca gaacacaggt aagtgccgtg tgtggttccc gcgggcctgg
     2221 cctctttacg ggttatggcc cttgcgtgcc ttgaattact tccacctggc tgcagtacgt
     2281 gattcttgat cccgagcttc gggttggaag tgggtgggag agttcgaggc cttgcgctta
     2341 aggagcccct tcgcctcgtg cttgagttga ggcctggcct gggcgctggg gccgccgcgt
     2401 gcgaatctgg tggcaccttc gcgcctgtct cgctgctttc gataagtctc tagccattta
     2461 aaatttttga tgacctgctg cgacgctttt tttctggcaa gatagtcttg taaatgcggg
     2521 ccaagatctg cacactggta tttcggtttt tggggccgcg ggcggcgacg gggcccgtgc
     2581 gtcccagcgc acatgttcgg cgaggcgggg cctgcgagcg cggccaccga gaatcggacg
     2641 ggggtagtct caagctggcc ggcctgctct ggtgcctggc ctcgcgccgc cgtgtatcgc
     2701 cccgccctgg gcggcaaggc tggcccggtc ggcaccagtt gcgtgagcgg aaagatggcc
     2761 gcttcccggc cctgctgcag ggagctcaaa atggaggacg cggcgctcgg gagagcgggc
     2821 gggtgagtca cccacacaaa ggaaaagggc ctttccgtcc tcagccgtcg cttcatgtga
     2881 ctccacggag taccgggcgc cgtccaggca cctcgattag ttctcgagct tttggagtac
     2941 gtcgtcttta ggttgggggg aggggtttta tgcgatggag tttccccaca ctgagtgggt
     3001 ggagactgaa gttaggccag cttggcactt gatgtaattc tccttggaat ttgccctttt
     3061 tgagtttgga tcttggttca ttctcaagcc tcagacagtg gttcaaagtt tttttcttcc
     3121 atttcaggtg tcgtgacgta cggccaccat ggaacgggtg ctcatgcacc ttagaaagtc
     3181 cagagccgat ctggaagctg aggccagggg cgagggcgaa accctggcta aacatagaaa
     3241 catcctgctg aagctggcca aagaaagaaa tctgaacatc gtgaagatca gagaggaagt
     3301 gggcatcagc ggcgagtctc tgatccacag acctgagatg ctggagacac tgaaggaggt
     3361 ggaagaggga ctgtacgacg ctgtgctgtg catggacatc gacagactgg gcagaggcaa
     3421 taccaaggag cagggcatta tcctggaggc cttcaagaat agcaacacca agatcatcac
     3481 ccctctgaaa acatacgacc tgaacaacga gttcgacgag gaatacgccg agtttggcgc
     3541 cttcatggcc aggcgggaat tcaagtttat caccagaaga ctgcagcggg gcagagtggc
     3601 cacagttgag gagggaaatt acatcgctcc aagaccacct tacggctaca tcatcgagaa
     3661 aaacaacaag gagcggtacc tgattcctca ccctgagcag gcccctatcg tgaagatgat
     3721 ctttgactgg tacacccacg acgatcctaa cgtgcggatg ggcgccagca agatcgccaa
     3781 cgagctgaac aagttctgca agagccctac aggcatcgcc tggaagggct ccaccgtgtt
     3841 gtctatcctg aagaatgccg tgtacgccgg cagaatccag tggcggaaga aggaggaaaa
     3901 aaaatctatc acaccaggca aaaaaaagga tgtccggatg cggcccaagg aacaatggat
     3961 cgacgtggag ggcaagcacg aacctctggt gtccatggaa acctacctga aggcccaaga
     4021 gatcctggag cggaagtacc acgtgcccta tcagatccag aacggcatca ctaaccccct
     4081 ggccggactg gtcaagtgcg gaatttgtgg tagcagcatg gtgtatagac cttacaccca
     4141 ccagcgccac ccccacctga tctgctacaa cagatactgt accaacaagt ctagcagatt
     4201 cgagtacgtg gaaagcaaaa ttatccaagg cctgcaccag ctgctggcac agtacaaggc
     4261 cgattggttc aaacatcaga gacccaaggt gaacgacgac agcgtggacc tgagacagaa
     4321 agccctccac cggctggaga aggaactgaa tgacctgtac aagcagaagg acaacctgca
     4381 caatctgctg gagaagggcg tgtacagcat cgatacattc ctggaaagaa gcaacatcct
     4441 ggctgaacgc atcgatgata ccaagaaggc catccacgac gccgagaagg ctctggccga
     4501 ggaggtgcag cggaacaaag tgaagaagga tatcatcccc acagtcgaga acgtgcttga
     4561 actgtactac aagacccagg accctgccaa gaagaacaac ctgctgaaga gcgtgctgga
     4621 ctacgctgtt tacagaaaag aaaagcacca gagagacgat gacttcacct tggtgcttta
     4681 tcctaagctg cctcagatca acagcggacc taagaaaaag aggaaggtgt ctggtagcga
     4741 gacacccggt accagtgaaa gcgccacgcc agaaagcagt gggagtgaga ctccgggtac
     4801 atctgaatca gcgacaccgg aatcaagtgg cggcagcagc ggcggcagca gcagccccaa
     4861 gaagaagaga aaggtggagg ccagcgacaa gaagtacagc atcggcctgg ccatcggcac
     4921 caactctgtg ggctgggccg tgatcaccga cgagtacaag gtgcccagca agaaattcaa
     4981 ggtgctgggc aacaccgacc ggcacagcat caagaagaac ctgatcggag ccctgctgtt
     5041 cgacagcggc gaaacagccg aggccacccg gctgaagaga accgccagaa gaagatacac
     5101 cagacggaag aaccggatct gctatctgca agagatcttc agcaacgaga tggccaaggt
     5161 ggacgacagc ttcttccaca gactggaaga gtccttcctg gtggaagagg ataagaagca
     5221 cgagcggcac cccatcttcg gcaacatcgt ggacgaggtg gcctaccacg agaagtaccc
     5281 caccatctac cacctgagaa agaaactggt ggacagcacc gacaaggccg acctgcggct
     5341 gatctatctg gccctggccc acatgatcaa gttccggggc cacttcctga tcgagggcga
     5401 cctgaacccc gacaacagcg acgtggacaa gctgttcatc cagctggtgc agacctacaa
     5461 ccagctgttc gaggaaaacc ccatcaacgc cagcggcgtg gacgccaagg ccatcctgtc
     5521 tgccagactg agcaagagca gacggctgga aaatctgatc gcccagctgc ccggcgagaa
     5581 gaagaatggc ctgttcggca acctgattgc cctgagcctg ggcctgaccc ccaacttcaa
     5641 gagcaacttc gacctggccg aggatgccaa actgcagctg agcaaggaca cctacgacga
     5701 cgacctggac aacctgctgg cccagatcgg cgaccagtac gccgacctgt ttctggccgc
     5761 caagaacctg tccgacgcca tcctgctgag cgacatcctg agagtgaaca ccgagatcac
     5821 caaggccccc ctgagcgcct ctatgatcaa gagatacgac gagcaccacc aggacctgac
     5881 cctgctgaaa gctctcgtgc ggcagcagct gcctgagaag tacaaagaga ttttcttcga
     5941 ccagagcaag aacggctacg ccggctacat tgacggcgga gccagccagg aagagttcta
     6001 caagttcatc aagcccatcc tggaaaagat ggacggcacc gaggaactgc tcgtgaagct
     6061 gaacagagag gacctgctgc ggaagcagcg gaccttcgac aacggcagca tcccccacca
     6121 gatccacctg ggagagctgc acgccattct gcggcggcag gaagattttt acccattcct
     6181 gaaggacaac cgggaaaaga tcgagaagat cctgaccttc cgcatcccct actacgtggg
     6241 ccctctggcc aggggaaaca gcagattcgc ctggatgacc agaaagagcg aggaaaccat
     6301 caccccctgg aacttcgagg aagtggtgga caagggcgct tccgcccaga gcttcatcga
     6361 gcggatgacc aacttcgata agaacctgcc caacgagaag gtgctgccca agcacagcct
     6421 gctgtacgag tacttcaccg tgtataacga gctgaccaaa gtgaaatacg tgaccgaggg
     6481 aatgagaaag cccgccttcc tgagcggcga gcagaaaaag gccatcgtgg acctgctgtt
     6541 caagaccaac cggaaagtga ccgtgaagca gctgaaagag gactacttca agaaaatcga
     6601 gtgcttcgac tccgtggaaa tctccggcgt ggaagatcgg ttcaacgcct ccctgggcac
     6661 ataccacgat ctgctgaaaa ttatcaagga caaggacttc ctggacaatg aggaaaacga
     6721 ggacattctg gaagatatcg tgctgaccct gacactgttt gaggacagag agatgatcga
     6781 ggaacggctg aaaacctatg cccacctgtt cgacgacaaa gtgatgaagc agctgaagcg
     6841 gcggagatac accggctggg gcaggctgag ccggaagctg atcaacggca tccgggacaa
     6901 gcagtccggc aagacaatcc tggatttcct gaagtccgac ggcttcgcca acagaaactt
     6961 catgcagctg atccacgacg acagcctgac ctttaaagag gacatccaga aagcccaggt
     7021 gtccggccag ggcgatagcc tgcacgagca cattgccaat ctggccggca gccccgccat
     7081 taagaagggc atcctgcaga cagtgaaggt ggtggacgag ctcgtgaaag tgatgggccg
     7141 gcacaagccc gagaacatcg tgatcgaaat ggccagagag aaccagacca cccagaaggg
     7201 acagaagaac agccgcgaga gaatgaagcg gatcgaagag ggcatcaaag agctgggcag
     7261 ccagatcctg aaagaacacc ccgtggaaaa cacccagctg cagaacgaga agctgtacct
     7321 gtactacctg cagaatgggc gggatatgta cgtggaccag gaactggaca tcaaccggct
     7381 gtccgactac gatgtggacg ctatcgtgcc tcagagcttt ctgaaggacg actccatcga
     7441 caacaaggtg ctgaccagaa gcgacaagaa ccggggcaag agcgacaacg tgccctccga
     7501 agaggtcgtg aagaagatga agaactactg gcggcagctg ctgaacgcca agctgattac
     7561 ccagagaaag ttcgacaatc tgaccaaggc cgagagaggc ggcctgagcg aactggataa
     7621 ggccggcttc atcaagagac agctggtgga aacccggcag atcacaaagc acgtggcaca
     7681 gatcctggac tcccggatga acactaagta cgacgagaat gacaagctga tccgggaagt
     7741 gaaagtgatc accctgaagt ccaagctggt gtccgatttc cggaaggatt tccagtttta
     7801 caaagtgcgc gagatcaaca actaccacca cgcccacgac gcctacctga acgccgtcgt
     7861 gggaaccgcc ctgatcaaaa agtaccctaa gctggaaagc gagttcgtgt acggcgacta
     7921 caaggtgtac gacgtgcgga agatgatcgc caagagcgag caggaaatcg gcaaggctac
     7981 cgccaagtac ttcttctaca gcaacatcat gaactttttc aagaccgaga ttaccctggc
     8041 caacggcgag atccggaagc ggcctctgat cgagacaaac ggcgaaaccg gggagatcgt
     8101 gtgggataag ggccgggatt ttgccaccgt gcggaaagtg ctgagcatgc cccaagtgaa
     8161 tatcgtgaaa aagaccgagg tgcagacagg cggcttcagc aaagagtcta tcctgcccaa
     8221 gaggaacagc gataagctga tcgccagaaa gaaggactgg gaccctaaga agtacggcgg
     8281 cttcgacagc cccaccgtgg cctattctgt gctggtggtg gccaaagtgg aaaagggcaa
     8341 gtccaagaaa ctgaagagtg tgaaagagct gctggggatc accatcatgg aaagaagcag
     8401 cttcgagaag aatcccatcg actttctgga agccaagggc tacaaagaag tgaaaaagga
     8461 cctgatcatc aagctgccta agtactccct gttcgagctg gaaaacggcc ggaagagaat
     8521 gctggcctct gccggcgaac tgcagaaggg aaacgaactg gccctgccct ccaaatatgt
     8581 gaacttcctg tacctggcca gccactatga gaagctgaag ggctcccccg aggataatga
     8641 gcagaaacag ctgtttgtgg aacagcacaa gcactacctg gacgagatca tcgagcagat
     8701 cagcgagttc tccaagagag tgatcctggc cgacgctaat ctggacaaag tgctgtccgc
     8761 ctacaacaag caccgggata agcccatcag agagcaggcc gagaatatca tccacctgtt
     8821 taccctgacc aatctgggag cccctgccgc cttcaagtac tttgacacca ccatcgaccg
     8881 gaagaggtac accagcacca aagaggtgct ggacgccacc ctgatccacc agagcatcac
     8941 cggcctgtac gagacacgga tcgacctgtc tcagctggga ggcgacggac ctaagaaaaa
     9001 gaggaaggtg gctagcggca gtggagaggg cagaggaagt ctgctaacat gcggtgacgt
     9061 cgaggagaat cctggcccag tgagcaaggg cgaggagctg ttcaccgggg tggtgcccat
     9121 cctggtcgag ctggacggcg acgtaaacgg ccacaagttc agcgtgtccg gcgagggcga
     9181 gggcgatgcc acctacggca agctgaccct gaagttcatc tgcaccaccg gcaagctgcc
     9241 cgtgccctgg cccaccctcg tgaccaccct gacctacggc gtgcagtgct tcagccgcta
     9301 ccccgaccac atgaagcagc acgacttctt caagtccgcc atgcccgaag gctacgtcca
     9361 ggagcgcacc atcttcttca aggacgacgg caactacaag acccgcgccg aggtgaagtt
     9421 cgagggcgac accctggtga accgcatcga gctgaagggc atcgacttca aggaggacgg
     9481 caacatcctg gggcacaagc tggagtacaa ctacaacagc cacaacgtct atatcatggc
     9541 cgacaagcag aagaacggca tcaaggtgaa cttcaagatc cgccacaaca tcgaggacgg
     9601 cagcgtgcag ctcgccgacc actaccagca gaacaccccc atcggcgacg gccccgtgct
     9661 gctgcccgac aaccactacc tgagcaccca gtccgccctg agcaaagacc ccaacgagaa
     9721 gcgcgatcac atggtcctgc tggagttcgt gaccgccgcc gggatcactc tcggcatgga
     9781 cgagctgtac aagtaagaat tcgatatcaa gcttatcgat aatcaacaac ttgtttattg
     9841 cagcttataa tggttacaaa taaagcaata gcatcacaaa tttcacaaat aaagcatttt
     9901 tttcactgca ttctagttgt ggtttgtcca aactcatcaa tgtatcttag acgtcaggtg
     9961 gcacttttcg gggaaatgtg cgcggaaccc ctatttgttt atttttctaa atacattcaa
    10021 atatgtatcc gctcatgaga caataaccct gataaatgct tcaataatat tgaaaaagga
    10081 agagtatgag tattcaacat ttccgtgtcg cccttattcc cttttttgcg gcattttgcc
    10141 ttcctgtttt tgctcaccca gaaacgctgg tgaaagtaaa agatgctgaa gatcagttgg
    10201 gtgcacgagt gggttacatc gaactggatc tcaacagcgg taagatcctt gagagttttc
    10261 gccccgaaga acgttttcca atgatgagca cttttaaagt tctgctatgt ggcgcggtat
    10321 tatcccgtat tgacgcc
//
