LOCUS       sequence-491688-       10337 bp    DNA     circular SYN 04-NOV-2025
DEFINITION  Mammalian expression of a human codon optimized engineered hifiDn29 
            recombinase fused to dCas9 and gRNA expression casette 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, Durrant MG, Wei 
            J, Pawluk A, Konermann S, Marson A, Gilbert LA, Hsu PD
  TITLE     Site-specific DNA insertion into the human genome with engineered 
            recombinases
  JOURNAL   bioRxiv 2024.11.01.621560
REFERENCE   2  (bases 1 to 10337)
  AUTHORS   .
  TITLE     Direct Submission
  JOURNAL   Exported Nov 4, 2025 from SnapGene Server 8.0.1
            https://www.snapgene.com
COMMENT     SGRef: number: 1; type: "Journal Article"; journalName: "bioRxiv 
            2024.11.01.621560"
COMMENT     Sequence Label: pEf1a-hifiDn29-dCas9-P2A-GFP
FEATURES             Location/Qualifiers
     source          1..10337
                     /mol_type="other DNA"
                     /organism="synthetic DNA construct"
     rep_origin      784..1372
                     /direction=RIGHT
                     /label=ori
                     /note="high-copy-number ColE1/pMB1/pBR322/pUC origin of 
                     replication"
     primer_bind     1273..1292
                     /label=pBR322ori-F
                     /note="pBR322 origin, forward primer"
     misc_RNA        complement(1505..1580)
                     /label=gRNA scaffold
                     /note="guide RNA scaffold for the Streptococcus pyogenes 
                     CRISPR/Cas9 system"
     promoter        complement(1601..1849)
                     /label=U6 promoter
                     /note="RNA polymerase III promoter for human U6 snRNA 
                     (Domitrovich & Kunkel, 2003)"
     primer_bind     complement(1659..1678)
                     /label=LKO.1 5'
                     /note="Human U6 promoter, forward primer"
     primer_bind     complement(1829..1849)
                     /label=hU6-F
                     /note="Human U6 promoter, forward primer"
     primer_bind     1900..1917
                     /label=L4440
                     /note="L4440 vector, forward primer"
     promoter        1962..3140
                     /label=EF-1-alpha promoter
                     /note="strong constitutive promoter for human elongation 
                     factor EF-1-alpha"
     intron          2193..3131
                     /label=EF-1-alpha intron A
                     /note="intron upstream of the start codon of human 
                     EF-1-alpha"
     primer_bind     3088..3108
                     /label=EF1a-F
                     /note="Human elongation factor-1a promoter, forward primer"
     regulatory      3147..3156
                     /label=Kozak sequence
                     /note="vertebrate consensus sequence for strong initiation 
                     of translation (Kozak, 1987)"
                     /regulatory_class="other"
     CDS             4713..4733
                     /codon_start=1
                     /product="nuclear localization signal of SV40 (simian virus
                     40) large T antigen"
                     /label=SV40 NLS
                     /translation="PKKKRKV"
     CDS             4860..4880
                     /codon_start=1
                     /product="nuclear localization signal of SV40 (simian virus
                     40) large T antigen"
                     /label=SV40 NLS
                     /translation="PKKKRKV"
     CDS             join(4890..4913,4914..4916,4917..7403,7404..7406,
                     7407..8990)
                     /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             4890..8990
                     /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             8994..9014
                     /codon_start=1
                     /product="nuclear localization signal of SV40 (simian virus
                     40) large T antigen"
                     /label=SV40 NLS
                     /translation="PKKKRKV"
     CDS             9030..9083
                     /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(9084..9086,9087..9800)
                     /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(9084..9086,9087..9797)
                     /codon_start=1
                     /product="enhanced GFP"
                     /label=EGFP
                     /translation="VSKGEELFTGVVPILVELDGDVNGHKFSVSGEGEGDATYGKLTLK
                     FICTTGKLPVPWPTLVTTLTYGVQCFSRYPDHMKQHDFFKSAMPEGYVQERTIFFKDDG
                     NYKTRAEVKFEGDTLVNRIELKGIDFKEDGNILGHKLEYNYNSHNVYIMADKQKNGIKV
                     NFKIRHNIEDGSVQLADHYQQNTPIGDGPVLLPDNHYLSTQSALSKDPNEKRDHMVLLE
                     FVTAAGITLGMDELYK"
     primer_bind     complement(9387..9406)
                     /label=EXFP-R
                     /note="For distinguishing EGFP variants, reverse primer"
     primer_bind     9734..9755
                     /label=EGFP-C
                     /note="EGFP, forward primer"
     polyA_signal    9832..9953
                     /label=SV40 poly(A) signal
                     /note="SV40 polyadenylation signal"
     primer_bind     complement(9869..9888)
                     /label=SV40pA-R
                     /note="SV40 polyA, reverse primer"
     primer_bind     9923..9942
                     /label=EBV-rev
                     /note="SV40 polyA terminator, reverse primer"
     promoter        9985..10089
                     /gene="bla"
                     /label=AmpR promoter
     CDS             join(10090..10158,10159..10337,1..613)
                     /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(10308..10327)
                     /label=Amp-R
                     /note="Ampicillin resistance gene, reverse primer"
ORIGIN
        1 cgccgggcaa gagcaactcg gtcgccgcat acactattct cagaatgact tggttgagta
       61 ctcaccagtc acagaaaagc atcttacgga tggcatgaca gtaagagaat tatgcagtgc
      121 tgccataacc atgagtgata acactgcggc caacttactt ctgacaacga tcggaggacc
      181 gaaggagcta accgcttttt tgcacaacat gggggatcat gtaactcgcc ttgatcgttg
      241 ggaaccggag ctgaatgaag ccataccaaa cgacgagcgt gacaccacga tgcctgtagc
      301 aatggcaaca acgttgcgca aactattaac tggcgaacta cttactctag cttcccggca
      361 acaattaata gactggatgg aggcggataa agttgcagga ccacttctgc gctcggccct
      421 tccggctggc tggtttattg ctgataaatc tggagccggt gagcgtgggt ctcgcggtat
      481 cattgcagca ctggggccag atggtaagcc ctcccgtatc gtagttatct acacgacggg
      541 gagtcaggca actatggatg aacgaaatag acagatcgct gagataggtg cctcactgat
      601 taagcattgg taactgtcag accaagttta ctcatatata ctttagattg atttaaaact
      661 tcatttttaa tttaaaagga tctaggtgaa gatccttttt gataatctca tgaccaaaat
      721 cccttaacgt gagttttcgt tccactgagc gtcagacccc gtagaaaaga tcaaaggatc
      781 ttcttgagat cctttttttc tgcgcgtaat ctgctgcttg caaacaaaaa aaccaccgct
      841 accagcggtg gtttgtttgc cggatcaaga gctaccaact ctttttccga aggtaactgg
      901 cttcagcaga gcgcagatac caaatactgt tcttctagtg tagccgtagt taggccacca
      961 cttcaagaac tctgtagcac cgcctacata cctcgctctg ctaatcctgt taccagtggc
     1021 tgctgccagt ggcgataagt cgtgtcttac cgggttggac tcaagacgat agttaccgga
     1081 taaggcgcag cggtcgggct gaacgggggg ttcgtgcaca cagcccagct tggagcgaac
     1141 gacctacacc gaactgagat acctacagcg tgagctatga gaaagcgcca cgcttcccga
     1201 agggagaaag gcggacaggt atccggtaag cggcagggtc ggaacaggag agcgcacgag
     1261 ggagcttcca gggggaaacg cctggtatct ttatagtcct gtcgggtttc gccacctctg
     1321 acttgagcgt cgatttttgt gatgctcgtc aggggggcgg agcctatgga aaaacgccag
     1381 caacgcggcc tttttacggt tcctggcctt ttgctggcct tttgctcaca tgttctttcc
     1441 tgcgttatcc cctgattctg tggataaccg tattaccgcc tttgcgcatc agaattcaaa
     1501 aaaagcaccg actcggtgcc actttttcaa gttgataacg gactagcctt attttaactt
     1561 gctatttcta gctctaaaac acaacagcag aatacacact ggtgtttcgt cctttccaca
     1621 agatatataa agccaagaaa tcgaaatact ttcaagttac ggtaagcata tgatagtcca
     1681 ttttaaaaca taattttaaa actgcaaact acccaagaaa ttattacttt ctacgtcacg
     1741 tattttgtac taatatcttt gtgtttacag tcaaattaat tccaattatc tctctaacag
     1801 ccttgtatcg tatatgcaaa tatgaaggaa tcatgggaaa taggccctcg ctagctgaag
     1861 tgagctgata ccgctcgccg cagccgaacg accgagcgca gcgagtcagt gagcgaggaa
     1921 gggaagagcg gaccttctag gtcttgaaag gagtgggaat tggctccggt gcccgtcagt
     1981 gggcagagcg cacatcgccc acagtccccg agaagttggg gggaggggtc ggcaattgaa
     2041 ccggtgccta gagaaggtgg cgcggggtaa actgggaaag tgatgtcgtg tactggctcc
     2101 gcctttttcc cgagggtggg ggagaaccgt atataagtgc agtagtcgcc gtgaacgttc
     2161 tttttcgcaa cgggtttgcc gccagaacac aggtaagtgc cgtgtgtggt tcccgcgggc
     2221 ctggcctctt tacgggttat ggcccttgcg tgccttgaat tacttccacc tggctgcagt
     2281 acgtgattct tgatcccgag cttcgggttg gaagtgggtg ggagagttcg aggccttgcg
     2341 cttaaggagc cccttcgcct cgtgcttgag ttgaggcctg gcctgggcgc tggggccgcc
     2401 gcgtgcgaat ctggtggcac cttcgcgcct gtctcgctgc tttcgataag tctctagcca
     2461 tttaaaattt ttgatgacct gctgcgacgc tttttttctg gcaagatagt cttgtaaatg
     2521 cgggccaaga tctgcacact ggtatttcgg tttttggggc cgcgggcggc gacggggccc
     2581 gtgcgtccca gcgcacatgt tcggcgaggc ggggcctgcg agcgcggcca ccgagaatcg
     2641 gacgggggta gtctcaagct ggccggcctg ctctggtgcc tggcctcgcg ccgccgtgta
     2701 tcgccccgcc ctgggcggca aggctggccc ggtcggcacc agttgcgtga gcggaaagat
     2761 ggccgcttcc cggccctgct gcagggagct caaaatggag gacgcggcgc tcgggagagc
     2821 gggcgggtga gtcacccaca caaaggaaaa gggcctttcc gtcctcagcc gtcgcttcat
     2881 gtgactccac ggagtaccgg gcgccgtcca ggcacctcga ttagttctcg agcttttgga
     2941 gtacgtcgtc tttaggttgg ggggaggggt tttatgcgat ggagtttccc cacactgagt
     3001 gggtggagac tgaagttagg ccagcttggc acttgatgta attctccttg gaatttgccc
     3061 tttttgagtt tggatcttgg ttcattctca agcctcagac agtggttcaa agtttttttc
     3121 ttccatttca ggtgtcgtga cgtacggcca ccatggaacg ggtgctcata caccttagaa
     3181 agtccagagc cgatctggaa gctgaggcca ggggcgaggg cgaaaccctg gctaaacata
     3241 gaaacatcct gctgaagctg gccaaagaaa gaaatctgaa catcgtgaag atcagagagg
     3301 aagtgggcat cagcggcgag tctctgatcc acagacctga gatgctggag acactgaagg
     3361 gggtggaaga gggactgtac gacgctgtgc tgtgcatgga catcgacaga ctgggcagag
     3421 gcaataccaa ggagcagggc attgtgctgg aggccttcaa gaatagcaac accaagatca
     3481 tcacccctct gaaaacatac gacctgaaca acgagttcga cgaggaatac gccgagtttg
     3541 gcgccttcat ggccaggcgg gaattcaagt ttatcaccag aagactgcag cggggcagag
     3601 tggccacagt tgaggaggga aattacatcg ctccaagacc accttacggc tacatcatcg
     3661 agaaaaacaa caaggagcgg tacctgattc ctcaccctga gcaggcccct atcgtgaaga
     3721 tgatctttga ctggtacacc cacgacgatc ctaacgtgcg gatgggcgcc agcaagatcg
     3781 ccaacgagct gaacaagttc tgcaagagcc ctacaggcat cccctggaag gtctccaccg
     3841 tgttgtctaa gttgaagaat gccgtgtacg ccggcagaat ccagtggcgg aagcgggagg
     3901 aaaaaaaatc tatcacacca ggcaaaaaaa aggatgtccg gatgcggccc aaggaacaat
     3961 ggatcgacgt ggagggcaag cacgaacctc tggtgtccat ggaaacctac ctgaaggccc
     4021 aagagatcct ggagcggaag taccacgtgc cctatcagaa gcagaacggc atcactaacc
     4081 ccctggccgg actggtcaag tgcgggattt gtggtagcag catggtgtat agaccttaca
     4141 cccacaagcg ccacccccac ctgatctgct acaaaagata ctgtaccaac aagtctagca
     4201 gattcgagta cgtggaaagc aaaattatcc aaggcctgca ccagctgctg gcacagtaca
     4261 aggccgattg gttcaaacat cagagaccca aggtgaacga cgacagcgtg gacctgagac
     4321 agaaagcccc ccaccggctg gagaaggaac tgaatgacct gtacaagcag aaggacaacc
     4381 tgcacaatct gctggagaag ggcgtgtaca gcatcgatac attcctggaa agaagcaaca
     4441 tcctggctga acgcatcgat gataccaaga aggccatcca cgacgccgag aaggctctgg
     4501 ccgaggaggt gcagcggaac aaagtgaaga aggatatcat ccccacagtc gagaacgtgc
     4561 ttgaactgta ctacaagacc caggaccctg ccaagaagaa caacctgctg aagagcgtgc
     4621 tggactacgc tgtttacaga aaagaaaagc accagagaaa cgatgacttc accttggtgc
     4681 tttatcctaa gctgcctcag atcaacagcg gacctaagaa aaagaggaag gtgtctggta
     4741 gcgagacacc cggtaccagt gaaagcgcca cgccagaaag cagtgggagt gagactccgg
     4801 gtacatctga atcagcgaca ccggaatcaa gtggcggcag cagcggcggc agcagcagcc
     4861 ccaagaagaa gagaaaggtg gaggccagcg acaagaagta cagcatcggc ctggccatcg
     4921 gcaccaactc tgtgggctgg gccgtgatca ccgacgagta caaggtgccc agcaagaaat
     4981 tcaaggtgct gggcaacacc gaccggcaca gcatcaagaa gaacctgatc ggagccctgc
     5041 tgttcgacag cggcgaaaca gccgaggcca cccggctgaa gagaaccgcc agaagaagat
     5101 acaccagacg gaagaaccgg atctgctatc tgcaagagat cttcagcaac gagatggcca
     5161 aggtggacga cagcttcttc cacagactgg aagagtcctt cctggtggaa gaggataaga
     5221 agcacgagcg gcaccccatc ttcggcaaca tcgtggacga ggtggcctac cacgagaagt
     5281 accccaccat ctaccacctg agaaagaaac tggtggacag caccgacaag gccgacctgc
     5341 ggctgatcta tctggccctg gcccacatga tcaagttccg gggccacttc ctgatcgagg
     5401 gcgacctgaa ccccgacaac agcgacgtgg acaagctgtt catccagctg gtgcagacct
     5461 acaaccagct gttcgaggaa aaccccatca acgccagcgg cgtggacgcc aaggccatcc
     5521 tgtctgccag actgagcaag agcagacggc tggaaaatct gatcgcccag ctgcccggcg
     5581 agaagaagaa tggcctgttc ggcaacctga ttgccctgag cctgggcctg acccccaact
     5641 tcaagagcaa cttcgacctg gccgaggatg ccaaactgca gctgagcaag gacacctacg
     5701 acgacgacct ggacaacctg ctggcccaga tcggcgacca gtacgccgac ctgtttctgg
     5761 ccgccaagaa cctgtccgac gccatcctgc tgagcgacat cctgagagtg aacaccgaga
     5821 tcaccaaggc ccccctgagc gcctctatga tcaagagata cgacgagcac caccaggacc
     5881 tgaccctgct gaaagctctc gtgcggcagc agctgcctga gaagtacaaa gagattttct
     5941 tcgaccagag caagaacggc tacgccggct acattgacgg cggagccagc caggaagagt
     6001 tctacaagtt catcaagccc atcctggaaa agatggacgg caccgaggaa ctgctcgtga
     6061 agctgaacag agaggacctg ctgcggaagc agcggacctt cgacaacggc agcatccccc
     6121 accagatcca cctgggagag ctgcacgcca ttctgcggcg gcaggaagat ttttacccat
     6181 tcctgaagga caaccgggaa aagatcgaga agatcctgac cttccgcatc ccctactacg
     6241 tgggccctct ggccagggga aacagcagat tcgcctggat gaccagaaag agcgaggaaa
     6301 ccatcacccc ctggaacttc gaggaagtgg tggacaaggg cgcttccgcc cagagcttca
     6361 tcgagcggat gaccaacttc gataagaacc tgcccaacga gaaggtgctg cccaagcaca
     6421 gcctgctgta cgagtacttc accgtgtata acgagctgac caaagtgaaa tacgtgaccg
     6481 agggaatgag aaagcccgcc ttcctgagcg gcgagcagaa aaaggccatc gtggacctgc
     6541 tgttcaagac caaccggaaa gtgaccgtga agcagctgaa agaggactac ttcaagaaaa
     6601 tcgagtgctt cgactccgtg gaaatctccg gcgtggaaga tcggttcaac gcctccctgg
     6661 gcacatacca cgatctgctg aaaattatca aggacaagga cttcctggac aatgaggaaa
     6721 acgaggacat tctggaagat atcgtgctga ccctgacact gtttgaggac agagagatga
     6781 tcgaggaacg gctgaaaacc tatgcccacc tgttcgacga caaagtgatg aagcagctga
     6841 agcggcggag atacaccggc tggggcaggc tgagccggaa gctgatcaac ggcatccggg
     6901 acaagcagtc cggcaagaca atcctggatt tcctgaagtc cgacggcttc gccaacagaa
     6961 acttcatgca gctgatccac gacgacagcc tgacctttaa agaggacatc cagaaagccc
     7021 aggtgtccgg ccagggcgat agcctgcacg agcacattgc caatctggcc ggcagccccg
     7081 ccattaagaa gggcatcctg cagacagtga aggtggtgga cgagctcgtg aaagtgatgg
     7141 gccggcacaa gcccgagaac atcgtgatcg aaatggccag agagaaccag accacccaga
     7201 agggacagaa gaacagccgc gagagaatga agcggatcga agagggcatc aaagagctgg
     7261 gcagccagat cctgaaagaa caccccgtgg aaaacaccca gctgcagaac gagaagctgt
     7321 acctgtacta cctgcagaat gggcgggata tgtacgtgga ccaggaactg gacatcaacc
     7381 ggctgtccga ctacgatgtg gacgctatcg tgcctcagag ctttctgaag gacgactcca
     7441 tcgacaacaa ggtgctgacc agaagcgaca agaaccgggg caagagcgac aacgtgccct
     7501 ccgaagaggt cgtgaagaag atgaagaact actggcggca gctgctgaac gccaagctga
     7561 ttacccagag aaagttcgac aatctgacca aggccgagag aggcggcctg agcgaactgg
     7621 ataaggccgg cttcatcaag agacagctgg tggaaacccg gcagatcaca aagcacgtgg
     7681 cacagatcct ggactcccgg atgaacacta agtacgacga gaatgacaag ctgatccggg
     7741 aagtgaaagt gatcaccctg aagtccaagc tggtgtccga tttccggaag gatttccagt
     7801 tttacaaagt gcgcgagatc aacaactacc accacgccca cgacgcctac ctgaacgccg
     7861 tcgtgggaac cgccctgatc aaaaagtacc ctaagctgga aagcgagttc gtgtacggcg
     7921 actacaaggt gtacgacgtg cggaagatga tcgccaagag cgagcaggaa atcggcaagg
     7981 ctaccgccaa gtacttcttc tacagcaaca tcatgaactt tttcaagacc gagattaccc
     8041 tggccaacgg cgagatccgg aagcggcctc tgatcgagac aaacggcgaa accggggaga
     8101 tcgtgtggga taagggccgg gattttgcca ccgtgcggaa agtgctgagc atgccccaag
     8161 tgaatatcgt gaaaaagacc gaggtgcaga caggcggctt cagcaaagag tctatcctgc
     8221 ccaagaggaa cagcgataag ctgatcgcca gaaagaagga ctgggaccct aagaagtacg
     8281 gcggcttcga cagccccacc gtggcctatt ctgtgctggt ggtggccaaa gtggaaaagg
     8341 gcaagtccaa gaaactgaag agtgtgaaag agctgctggg gatcaccatc atggaaagaa
     8401 gcagcttcga gaagaatccc atcgactttc tggaagccaa gggctacaaa gaagtgaaaa
     8461 aggacctgat catcaagctg cctaagtact ccctgttcga gctggaaaac ggccggaaga
     8521 gaatgctggc ctctgccggc gaactgcaga agggaaacga actggccctg ccctccaaat
     8581 atgtgaactt cctgtacctg gccagccact atgagaagct gaagggctcc cccgaggata
     8641 atgagcagaa acagctgttt gtggaacagc acaagcacta cctggacgag atcatcgagc
     8701 agatcagcga gttctccaag agagtgatcc tggccgacgc taatctggac aaagtgctgt
     8761 ccgcctacaa caagcaccgg gataagccca tcagagagca ggccgagaat atcatccacc
     8821 tgtttaccct gaccaatctg ggagcccctg ccgccttcaa gtactttgac accaccatcg
     8881 accggaagag gtacaccagc accaaagagg tgctggacgc caccctgatc caccagagca
     8941 tcaccggcct gtacgagaca cggatcgacc tgtctcagct gggaggcgac ggacctaaga
     9001 aaaagaggaa ggtggctagc ggcagtggag agggcagagg aagtctgcta acatgcggtg
     9061 acgtcgagga gaatcctggc ccagtgagca agggcgagga gctgttcacc ggggtggtgc
     9121 ccatcctggt cgagctggac ggcgacgtaa acggccacaa gttcagcgtg tccggcgagg
     9181 gcgagggcga tgccacctac ggcaagctga ccctgaagtt catctgcacc accggcaagc
     9241 tgcccgtgcc ctggcccacc ctcgtgacca ccctgaccta cggcgtgcag tgcttcagcc
     9301 gctaccccga ccacatgaag cagcacgact tcttcaagtc cgccatgccc gaaggctacg
     9361 tccaggagcg caccatcttc ttcaaggacg acggcaacta caagacccgc gccgaggtga
     9421 agttcgaggg cgacaccctg gtgaaccgca tcgagctgaa gggcatcgac ttcaaggagg
     9481 acggcaacat cctggggcac aagctggagt acaactacaa cagccacaac gtctatatca
     9541 tggccgacaa gcagaagaac ggcatcaagg tgaacttcaa gatccgccac aacatcgagg
     9601 acggcagcgt gcagctcgcc gaccactacc agcagaacac ccccatcggc gacggccccg
     9661 tgctgctgcc cgacaaccac tacctgagca cccagtccgc cctgagcaaa gaccccaacg
     9721 agaagcgcga tcacatggtc ctgctggagt tcgtgaccgc cgccgggatc actctcggca
     9781 tggacgagct gtacaagtaa gaattcgata tcaagcttat cgataatcaa caacttgttt
     9841 attgcagctt ataatggtta caaataaagc aatagcatca caaatttcac aaataaagca
     9901 tttttttcac tgcattctag ttgtggtttg tccaaactca tcaatgtatc ttagacgtca
     9961 ggtggcactt ttcggggaaa tgtgcgcgga acccctattt gtttattttt ctaaatacat
    10021 tcaaatatgt atccgctcat gagacaataa ccctgataaa tgcttcaata atattgaaaa
    10081 aggaagagta tgagtattca acatttccgt gtcgccctta ttcccttttt tgcggcattt
    10141 tgccttcctg tttttgctca cccagaaacg ctggtgaaag taaaagatgc tgaagatcag
    10201 ttgggtgcac gagtgggtta catcgaactg gatctcaaca gcggtaagat ccttgagagt
    10261 tttcgccccg aagaacgttt tccaatgatg agcactttta aagttctgct atgtggcgcg
    10321 gtattatccc gtattga
//
