LOCUS       sequence-223636-       10118 bp    DNA     circular SYN 15-DEC-2025
DEFINITION  Cas9 GFP plasmid constitutively expressed from CAG promoter. AAV 
            repeat region removed to increase expression.
ACCESSION   .
VERSION     .
KEYWORDS    .
SOURCE      synthetic DNA construct
  ORGANISM  synthetic DNA construct
REFERENCE   1  (bases 1 to 10118)
  AUTHORS   Hojland Knudsen C, Asgrimsdottir ES, Rahimi K, Gill KP, Frandsen S, 
            Hvolbol Buchholdt S, Chen M, Kjems J, Febbraro F, Denham M
  TITLE     A Modified Monomeric Red Fluorescent Protein Reporter for Assessing 
            CRISPR Activity.
  JOURNAL   Front Cell Dev Biol. 2018 May 15;6:54. doi: 
            10.3389/fcell.2018.00054. eCollection 2018.
  PUBMED    29868584
REFERENCE   2  (bases 1 to 10118)
  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: "Front Cell 
            Dev Biol."; date: "2018-05-15"; volume: "6:54. doi"; pages: " 
            10.3389/fcell.2018.00054. eCollection 2018"
COMMENT     Sequence Label: pCAG-SpCas9-2A-GFP-noITR
FEATURES             Location/Qualifiers
     source          1..10118
                     /mol_type="other DNA"
                     /organism="synthetic DNA construct"
     promoter        6..254
                     /label=U6 promoter
                     /note="RNA polymerase III promoter for human U6 snRNA 
                     (Domitrovich & Kunkel, 2003)"
     primer_bind     6..26
                     /label=hU6-F
                     /note="Human U6 promoter, forward primer"
     primer_bind     177..196
                     /label=LKO.1 5'
                     /note="Human U6 promoter, forward primer"
     misc_RNA        273..348
                     /label=gRNA scaffold
                     /note="guide RNA scaffold for the Streptococcus pyogenes 
                     CRISPR/Cas9 system"
     enhancer        465..844
                     /label=CMV enhancer
                     /note="human cytomegalovirus immediate early enhancer"
     promoter        847..1122
                     /label=chicken beta-actin promoter
     intron          1123..2131
                     /label=chimeric intron
                     /note="chimera between introns from chicken beta-actin and 
                     rabbit beta-globin"
     primer_bind     2055..2077
                     /label=pCAGGS-5
                     /note="Chimeric intron in CAG promoter, forward primer"
     regulatory      2198..2207
                     /label=Kozak sequence
                     /note="vertebrate consensus sequence for strong initiation 
                     of translation (Kozak, 1987)"
                     /regulatory_class="other"
     CDS             2207..2272
                     /codon_start=1
                     /product="three tandem FLAG(R) epitope tags, followed by an
                     enterokinase cleavage site"
                     /label=3xFLAG
                     /translation="DYKDHDGDYKDHDIDYKDDDDK"
     CDS             2279..2299
                     /codon_start=1
                     /product="nuclear localization signal of SV40 (simian virus
                     40) large T antigen"
                     /label=SV40 NLS
                     /translation="PKKKRKV"
     CDS             2324..6424
                     /codon_start=1
                     /product="Cas9 (Csn1) endonuclease from the Streptococcus 
                     pyogenes Type II CRISPR/Cas system"
                     /label=Cas9
                     /note="generates RNA-guided double strand breaks in DNA"
                     /translation="DKKYSIGLDIGTNSVGWAVITDEYKVPSKKFKVLGNTDRHSIKKN
                     LIGALLFDSGETAEATRLKRTARRRYTRRKNRICYLQEIFSNEMAKVDDSFFHRLEESF
                     LVEEDKKHERHPIFGNIVDEVAYHEKYPTIYHLRKKLVDSTDKADLRLIYLALAHMIKF
                     RGHFLIEGDLNPDNSDVDKLFIQLVQTYNQLFEENPINASGVDAKAILSARLSKSRRLE
                     NLIAQLPGEKKNGLFGNLIALSLGLTPNFKSNFDLAEDAKLQLSKDTYDDDLDNLLAQI
                     GDQYADLFLAAKNLSDAILLSDILRVNTEITKAPLSASMIKRYDEHHQDLTLLKALVRQ
                     QLPEKYKEIFFDQSKNGYAGYIDGGASQEEFYKFIKPILEKMDGTEELLVKLNREDLLR
                     KQRTFDNGSIPHQIHLGELHAILRRQEDFYPFLKDNREKIEKILTFRIPYYVGPLARGN
                     SRFAWMTRKSEETITPWNFEEVVDKGASAQSFIERMTNFDKNLPNEKVLPKHSLLYEYF
                     TVYNELTKVKYVTEGMRKPAFLSGEQKKAIVDLLFKTNRKVTVKQLKEDYFKKIECFDS
                     VEISGVEDRFNASLGTYHDLLKIIKDKDFLDNEENEDILEDIVLTLTLFEDREMIEERL
                     KTYAHLFDDKVMKQLKRRRYTGWGRLSRKLINGIRDKQSGKTILDFLKSDGFANRNFMQ
                     LIHDDSLTFKEDIQKAQVSGQGDSLHEHIANLAGSPAIKKGILQTVKVVDELVKVMGRH
                     KPENIVIEMARENQTTQKGQKNSRERMKRIEEGIKELGSQILKEHPVENTQLQNEKLYL
                     YYLQNGRDMYVDQELDINRLSDYDVDHIVPQSFLKDDSIDNKVLTRSDKNRGKSDNVPS
                     EEVVKKMKNYWRQLLNAKLITQRKFDNLTKAERGGLSELDKAGFIKRQLVETRQITKHV
                     AQILDSRMNTKYDENDKLIREVKVITLKSKLVSDFRKDFQFYKVREINNYHHAHDAYLN
                     AVVGTALIKKYPKLESEFVYGDYKVYDVRKMIAKSEQEIGKATAKYFFYSNIMNFFKTE
                     ITLANGEIRKRPLIETNGETGEIVWDKGRDFATVRKVLSMPQVNIVKKTEVQTGGFSKE
                     SILPKRNSDKLIARKKDWDPKKYGGFDSPTVAYSVLVVAKVEKGKSKKLKSVKELLGIT
                     IMERSSFEKNPIDFLEAKGYKEVKKDLIIKLPKYSLFELENGRKRMLASAGELQKGNEL
                     ALPSKYVNFLYLASHYEKLKGSPEDNEQKQLFVEQHKHYLDEIIEQISEFSKRVILADA
                     NLDKVLSAYNKHRDKPIREQAENIIHLFTLTNLGAPAAFKYFDTTIDRKRYTSTKEVLD
                     ATLIHQSITGLYETRIDLSQLGGD"
     CDS             2324..6424
                     /codon_start=1
                     /product="Cas9 (Csn1) endonuclease from the Streptococcus 
                     pyogenes Type II CRISPR/Cas system"
                     /label=Cas9
                     /note="generates RNA-guided double strand breaks in DNA"
                     /translation="DKKYSIGLDIGTNSVGWAVITDEYKVPSKKFKVLGNTDRHSIKKN
                     LIGALLFDSGETAEATRLKRTARRRYTRRKNRICYLQEIFSNEMAKVDDSFFHRLEESF
                     LVEEDKKHERHPIFGNIVDEVAYHEKYPTIYHLRKKLVDSTDKADLRLIYLALAHMIKF
                     RGHFLIEGDLNPDNSDVDKLFIQLVQTYNQLFEENPINASGVDAKAILSARLSKSRRLE
                     NLIAQLPGEKKNGLFGNLIALSLGLTPNFKSNFDLAEDAKLQLSKDTYDDDLDNLLAQI
                     GDQYADLFLAAKNLSDAILLSDILRVNTEITKAPLSASMIKRYDEHHQDLTLLKALVRQ
                     QLPEKYKEIFFDQSKNGYAGYIDGGASQEEFYKFIKPILEKMDGTEELLVKLNREDLLR
                     KQRTFDNGSIPHQIHLGELHAILRRQEDFYPFLKDNREKIEKILTFRIPYYVGPLARGN
                     SRFAWMTRKSEETITPWNFEEVVDKGASAQSFIERMTNFDKNLPNEKVLPKHSLLYEYF
                     TVYNELTKVKYVTEGMRKPAFLSGEQKKAIVDLLFKTNRKVTVKQLKEDYFKKIECFDS
                     VEISGVEDRFNASLGTYHDLLKIIKDKDFLDNEENEDILEDIVLTLTLFEDREMIEERL
                     KTYAHLFDDKVMKQLKRRRYTGWGRLSRKLINGIRDKQSGKTILDFLKSDGFANRNFMQ
                     LIHDDSLTFKEDIQKAQVSGQGDSLHEHIANLAGSPAIKKGILQTVKVVDELVKVMGRH
                     KPENIVIEMARENQTTQKGQKNSRERMKRIEEGIKELGSQILKEHPVENTQLQNEKLYL
                     YYLQNGRDMYVDQELDINRLSDYDVDHIVPQSFLKDDSIDNKVLTRSDKNRGKSDNVPS
                     EEVVKKMKNYWRQLLNAKLITQRKFDNLTKAERGGLSELDKAGFIKRQLVETRQITKHV
                     AQILDSRMNTKYDENDKLIREVKVITLKSKLVSDFRKDFQFYKVREINNYHHAHDAYLN
                     AVVGTALIKKYPKLESEFVYGDYKVYDVRKMIAKSEQEIGKATAKYFFYSNIMNFFKTE
                     ITLANGEIRKRPLIETNGETGEIVWDKGRDFATVRKVLSMPQVNIVKKTEVQTGGFSKE
                     SILPKRNSDKLIARKKDWDPKKYGGFDSPTVAYSVLVVAKVEKGKSKKLKSVKELLGIT
                     IMERSSFEKNPIDFLEAKGYKEVKKDLIIKLPKYSLFELENGRKRMLASAGELQKGNEL
                     ALPSKYVNFLYLASHYEKLKGSPEDNEQKQLFVEQHKHYLDEIIEQISEFSKRVILADA
                     NLDKVLSAYNKHRDKPIREQAENIIHLFTLTNLGAPAAFKYFDTTIDRKRYTSTKEVLD
                     ATLIHQSITGLYETRIDLSQLGGD"
     CDS             6425..6472
                     /codon_start=1
                     /product="bipartite nuclear localization signal from 
                     nucleoplasmin"
                     /label=nucleoplasmin NLS
                     /translation="KRPAATKKAGQAKKKK"
     CDS             6488..6541
                     /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(6542..6544,6545..7255)
                     /codon_start=1
                     /product="enhanced GFP"
                     /label=EGFP
                     /translation="VSKGEELFTGVVPILVELDGDVNGHKFSVSGEGEGDATYGKLTLK
                     FICTTGKLPVPWPTLVTTLTYGVQCFSRYPDHMKQHDFFKSAMPEGYVQERTIFFKDDG
                     NYKTRAEVKFEGDTLVNRIELKGIDFKEDGNILGHKLEYNYNSHNVYIMADKQKNGIKV
                     NFKIRHNIEDGSVQLADHYQQNTPIGDGPVLLPDNHYLSTQSALSKDPNEKRDHMVLLE
                     FVTAAGITLGMDELYK"
     CDS             join(6542..6544,6545..7255)
                     /codon_start=1
                     /product="the original enhanced GFP (Yang et al., 1996)"
                     /label=EGFP
                     /note="mammalian codon-optimized"
                     /translation="VSKGEELFTGVVPILVELDGDVNGHKFSVSGEGEGDATYGKLTLK
                     FICTTGKLPVPWPTLVTTLTYGVQCFSRYPDHMKQHDFFKSAMPEGYVQERTIFFKDDG
                     NYKTRAEVKFEGDTLVNRIELKGIDFKEDGNILGHKLEYNYNSHNVYIMADKQKNGIKV
                     NFKIRHNIEDGSVQLADHYQQNTPIGDGPVLLPDNHYLSTQSALSKDPNEKRDHMVLLE
                     FVTAAGITLGMDELYK"
     primer_bind     complement(6845..6864)
                     /label=EXFP-R
                     /note="For distinguishing EGFP variants, reverse primer"
     primer_bind     7192..7213
                     /label=EGFP-C
                     /note="EGFP, forward primer"
     primer_bind     complement(7283..7300)
                     /label=BGH-rev
                     /note="Bovine growth hormone terminator, reverse primer. 
                     Also called BGH reverse"
     polyA_signal    7289..7496
                     /label=bGH poly(A) signal
                     /note="bovine growth hormone polyadenylation signal"
     rep_origin      7601..8056
                     /direction=RIGHT
                     /label=f1 ori
                     /note="f1 bacteriophage origin of replication; arrow 
                     indicates direction of (+) strand synthesis"
     primer_bind     complement(7688..7707)
                     /label=F1ori-R
                     /note="F1 origin, reverse primer"
     primer_bind     7898..7919
                     /label=F1ori-F
                     /note="F1 origin, forward primer"
     primer_bind     complement(8073..8092)
                     /label=pRS-marker
                     /note="pRS vectors, use to sequence yeast selectable 
                     marker"
     primer_bind     8192..8214
                     /label=pGEX 3'
                     /note="pGEX vectors, reverse primer"
     primer_bind     complement(8252..8270)
                     /label=pBRforEco
                     /note="pBR322 vectors, upsteam of EcoRI site, forward 
                     primer"
     promoter        8338..8442
                     /gene="bla"
                     /label=AmpR promoter
     CDS             join(8443..8511,8512..9303)
                     /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(8661..8680)
                     /label=Amp-R
                     /note="Ampicillin resistance gene, reverse primer"
     rep_origin      9474..10062
                     /direction=RIGHT
                     /label=ori
                     /note="high-copy-number ColE1/pMB1/pBR322/pUC origin of 
                     replication"
     primer_bind     9963..9982
                     /label=pBR322ori-F
                     /note="pBR322 origin, forward primer"
ORIGIN
        1 catgtgaggg cctatttccc atgattcctt catatttgca tatacgatac aaggctgtta
       61 gagagataat tggaattaat ttgactgtaa acacaaagat attagtacaa aatacgtgac
      121 gtagaaagta ataatttctt gggtagtttg cagttttaaa attatgtttt aaaatggact
      181 atcatatgct taccgtaact tgaaagtatt tcgatttctt ggctttatat atcttgtgga
      241 aaggacgaaa caccgggtct tcgagaagac ctgttttaga gctagaaata gcaagttaaa
      301 ataaggctag tccgttatca acttgaaaaa gtggcaccga gtcggtgctt ttttgtttta
      361 gagctagaaa tagcaagtta aaataaggct agtccgtttt tagcgcgtgc gccaattctg
      421 cagacaaatg gctctagagg tacccgggga tctatatata tgtcgacatt gattattgac
      481 tagttattaa tagtaatcaa ttacggggtc attagttcat agcccatata tggagttccg
      541 cgttacataa cttacggtaa atggcccgcc tggctgaccg cccaacgacc cccgcccatt
      601 gacgtcaata atgacgtatg ttcccatagt aacgccaata gggactttcc attgacgtca
      661 atgggtggac tatttacggt aaactgccca cttggcagta catcaagtgt atcatatgcc
      721 aagtacgccc cctattgacg tcaatgacgg taaatggccc gcctggcatt atgcccagta
      781 catgacctta tgggactttc ctacttggca gtacatctac gtattagtca tcgctattac
      841 catgggtcga ggtgagcccc acgttctgct tcactctccc catctccccc ccctccccac
      901 ccccaatttt gtatttattt attttttaat tattttgtgc agcgatgggg gcgggggggg
      961 ggggggcgcg cgccaggcgg ggcggggcgg ggcgaggggc ggggcggggc gaggcggaga
     1021 ggtgcggcgg cagccaatca gagcggcgcg ctccgaaagt ttccttttat ggcgaggcgg
     1081 cggcggcggc ggccctataa aaagcgaagc gcgcggcggg cgggagtcgc tgcgttgcct
     1141 tcgccccgtg ccccgctccg cgccgcctcg cgccgcccgc cccggctctg actgaccgcg
     1201 ttactcccac aggtgagcgg gcgggacggc ccttctcctc cgggctgtaa ttagcgcttg
     1261 gtttaatgac ggctcgtttc ttttctgtgg ctgcgtgaaa gccttaaagg gctccgggag
     1321 ggccctttgt gcggggggga gcggctcggg gggtgcgtgc gtgtgtgtgt gcgtggggag
     1381 cgccgcgtgc ggcccgcgct gcccggcggc tgtgagcgct gcgggcgcgg cgcggggctt
     1441 tgtgcgctcc gcgtgtgcgc gaggggagcg cggccggggg cggtgccccg cggtgcgggg
     1501 gggctgcgag gggaacaaag gctgcgtgcg gggtgtgtgc gtgggggggt gagcaggggg
     1561 tgtgggcgcg gcggtcgggc tgtaaccccc ccctgcaccc ccctccccga gttgctgagc
     1621 acggcccggc ttcgggtgcg gggctccgtg cggggcgtgg cgcggggctc gccgtgccgg
     1681 gcggggggtg gcggcaggtg ggggtgccgg gcggggcggg gccgcctcgg gccggggagg
     1741 gctcggggga ggggcgcggc ggccccggag cgccggcggc tgtcgaggcg cggcgagccg
     1801 cagccattgc cttttatggt aatcgtgcga gagggcgcag ggacttcctt tgtcccaaat
     1861 ctggcggagc cgaaatctgg gaggcgccgc cgcaccccct ctagcgggcg cgggcgaagc
     1921 ggtgcggcgc cggcaggaag gaaatgggcg gggagggcct tcgtgcgtcg ccgcgccgcc
     1981 gtccccttct ccatctccag cctcggggct gccgcagggg gacggctgcc ttcggggggg
     2041 acggggcagg gcggggttcg gcttctggcg tgtgaccggc ggctctagag cctctgctaa
     2101 ccatgttcat gccttcttct ttttcctaca gctcctgggc aacgtgctgg ttgttgtgct
     2161 gtctcatcat tttggcaaag aattcctcga gaccggtgcc accatggact ataaggacca
     2221 cgacggagac tacaaggatc atgatattga ttacaaagac gatgacgata agatggcccc
     2281 aaagaagaag cggaaggtcg gtatccacgg agtcccagca gccgacaaga agtacagcat
     2341 cggcctggac atcggcacca actctgtggg ctgggccgtg atcaccgacg agtacaaggt
     2401 gcccagcaag aaattcaagg tgctgggcaa caccgaccgg cacagcatca agaagaacct
     2461 gatcggagcc ctgctgttcg acagcggcga aacagccgag gccacccggc tgaagagaac
     2521 cgccagaaga agatacacca gacggaagaa ccggatctgc tatctgcaag agatcttcag
     2581 caacgagatg gccaaggtgg acgacagctt cttccacaga ctggaagagt ccttcctggt
     2641 ggaagaggat aagaagcacg agcggcaccc catcttcggc aacatcgtgg acgaggtggc
     2701 ctaccacgag aagtacccca ccatctacca cctgagaaag aaactggtgg acagcaccga
     2761 caaggccgac ctgcggctga tctatctggc cctggcccac atgatcaagt tccggggcca
     2821 cttcctgatc gagggcgacc tgaaccccga caacagcgac gtggacaagc tgttcatcca
     2881 gctggtgcag acctacaacc agctgttcga ggaaaacccc atcaacgcca gcggcgtgga
     2941 cgccaaggcc atcctgtctg ccagactgag caagagcaga cggctggaaa atctgatcgc
     3001 ccagctgccc ggcgagaaga agaatggcct gttcggaaac ctgattgccc tgagcctggg
     3061 cctgaccccc aacttcaaga gcaacttcga cctggccgag gatgccaaac tgcagctgag
     3121 caaggacacc tacgacgacg acctggacaa cctgctggcc cagatcggcg accagtacgc
     3181 cgacctgttt ctggccgcca agaacctgtc cgacgccatc ctgctgagcg acatcctgag
     3241 agtgaacacc gagatcacca aggcccccct gagcgcctct atgatcaaga gatacgacga
     3301 gcaccaccag gacctgaccc tgctgaaagc tctcgtgcgg cagcagctgc ctgagaagta
     3361 caaagagatt ttcttcgacc agagcaagaa cggctacgcc ggctacattg acggcggagc
     3421 cagccaggaa gagttctaca agttcatcaa gcccatcctg gaaaagatgg acggcaccga
     3481 ggaactgctc gtgaagctga acagagagga cctgctgcgg aagcagcgga ccttcgacaa
     3541 cggcagcatc ccccaccaga tccacctggg agagctgcac gccattctgc ggcggcagga
     3601 agatttttac ccattcctga aggacaaccg ggaaaagatc gagaagatcc tgaccttccg
     3661 catcccctac tacgtgggcc ctctggccag gggaaacagc agattcgcct ggatgaccag
     3721 aaagagcgag gaaaccatca ccccctggaa cttcgaggaa gtggtggaca agggcgcttc
     3781 cgcccagagc ttcatcgagc ggatgaccaa cttcgataag aacctgccca acgagaaggt
     3841 gctgcccaag cacagcctgc tgtacgagta cttcaccgtg tataacgagc tgaccaaagt
     3901 gaaatacgtg accgagggaa tgagaaagcc cgccttcctg agcggcgagc agaaaaaggc
     3961 catcgtggac ctgctgttca agaccaaccg gaaagtgacc gtgaagcagc tgaaagagga
     4021 ctacttcaag aaaatcgagt gcttcgactc cgtggaaatc tccggcgtgg aagatcggtt
     4081 caacgcctcc ctgggcacat accacgatct gctgaaaatt atcaaggaca aggacttcct
     4141 ggacaatgag gaaaacgagg acattctgga agatatcgtg ctgaccctga cactgtttga
     4201 ggacagagag atgatcgagg aacggctgaa aacctatgcc cacctgttcg acgacaaagt
     4261 gatgaagcag ctgaagcggc ggagatacac cggctggggc aggctgagcc ggaagctgat
     4321 caacggcatc cgggacaagc agtccggcaa gacaatcctg gatttcctga agtccgacgg
     4381 cttcgccaac agaaacttca tgcagctgat ccacgacgac agcctgacct ttaaagagga
     4441 catccagaaa gcccaggtgt ccggccaggg cgatagcctg cacgagcaca ttgccaatct
     4501 ggccggcagc cccgccatta agaagggcat cctgcagaca gtgaaggtgg tggacgagct
     4561 cgtgaaagtg atgggccggc acaagcccga gaacatcgtg atcgaaatgg ccagagagaa
     4621 ccagaccacc cagaagggac agaagaacag ccgcgagaga atgaagcgga tcgaagaggg
     4681 catcaaagag ctgggcagcc agatcctgaa agaacacccc gtggaaaaca cccagctgca
     4741 gaacgagaag ctgtacctgt actacctgca gaatgggcgg gatatgtacg tggaccagga
     4801 actggacatc aaccggctgt ccgactacga tgtggaccat atcgtgcctc agagctttct
     4861 gaaggacgac tccatcgaca acaaggtgct gaccagaagc gacaagaacc ggggcaagag
     4921 cgacaacgtg ccctccgaag aggtcgtgaa gaagatgaag aactactggc ggcagctgct
     4981 gaacgccaag ctgattaccc agagaaagtt cgacaatctg accaaggccg agagaggcgg
     5041 cctgagcgaa ctggataagg ccggcttcat caagagacag ctggtggaaa cccggcagat
     5101 cacaaagcac gtggcacaga tcctggactc ccggatgaac actaagtacg acgagaatga
     5161 caagctgatc cgggaagtga aagtgatcac cctgaagtcc aagctggtgt ccgatttccg
     5221 gaaggatttc cagttttaca aagtgcgcga gatcaacaac taccaccacg cccacgacgc
     5281 ctacctgaac gccgtcgtgg gaaccgccct gatcaaaaag taccctaagc tggaaagcga
     5341 gttcgtgtac ggcgactaca aggtgtacga cgtgcggaag atgatcgcca agagcgagca
     5401 ggaaatcggc aaggctaccg ccaagtactt cttctacagc aacatcatga actttttcaa
     5461 gaccgagatt accctggcca acggcgagat ccggaagcgg cctctgatcg agacaaacgg
     5521 cgaaaccggg gagatcgtgt gggataaggg ccgggatttt gccaccgtgc ggaaagtgct
     5581 gagcatgccc caagtgaata tcgtgaaaaa gaccgaggtg cagacaggcg gcttcagcaa
     5641 agagtctatc ctgcccaaga ggaacagcga taagctgatc gccagaaaga aggactggga
     5701 ccctaagaag tacggcggct tcgacagccc caccgtggcc tattctgtgc tggtggtggc
     5761 caaagtggaa aagggcaagt ccaagaaact gaagagtgtg aaagagctgc tggggatcac
     5821 catcatggaa agaagcagct tcgagaagaa tcccatcgac tttctggaag ccaagggcta
     5881 caaagaagtg aaaaaggacc tgatcatcaa gctgcctaag tactccctgt tcgagctgga
     5941 aaacggccgg aagagaatgc tggcctctgc cggcgaactg cagaagggaa acgaactggc
     6001 cctgccctcc aaatatgtga acttcctgta cctggccagc cactatgaga agctgaaggg
     6061 ctcccccgag gataatgagc agaaacagct gtttgtggaa cagcacaagc actacctgga
     6121 cgagatcatc gagcagatca gcgagttctc caagagagtg atcctggccg acgctaatct
     6181 ggacaaagtg ctgtccgcct acaacaagca ccgggataag cccatcagag agcaggccga
     6241 gaatatcatc cacctgttta ccctgaccaa tctgggagcc cctgccgcct tcaagtactt
     6301 tgacaccacc atcgaccgga agaggtacac cagcaccaaa gaggtgctgg acgccaccct
     6361 gatccaccag agcatcaccg gcctgtacga gacacggatc gacctgtctc agctgggagg
     6421 cgacaaaagg ccggcggcca cgaaaaaggc cggccaggca aaaaagaaaa aggaattcgg
     6481 cagtggagag ggcagaggaa gtctgctaac atgcggtgac gtcgaggaga atcctggccc
     6541 agtgagcaag ggcgaggagc tgttcaccgg ggtggtgccc atcctggtcg agctggacgg
     6601 cgacgtaaac ggccacaagt tcagcgtgtc cggcgagggc gagggcgatg ccacctacgg
     6661 caagctgacc ctgaagttca tctgcaccac cggcaagctg cccgtgccct ggcccaccct
     6721 cgtgaccacc ctgacctacg gcgtgcagtg cttcagccgc taccccgacc acatgaagca
     6781 gcacgacttc ttcaagtccg ccatgcccga aggctacgtc caggagcgca ccatcttctt
     6841 caaggacgac ggcaactaca agacccgcgc cgaggtgaag ttcgagggcg acaccctggt
     6901 gaaccgcatc gagctgaagg gcatcgactt caaggaggac ggcaacatcc tggggcacaa
     6961 gctggagtac aactacaaca gccacaacgt ctatatcatg gccgacaagc agaagaacgg
     7021 catcaaggtg aacttcaaga tccgccacaa catcgaggac ggcagcgtgc agctcgccga
     7081 ccactaccag cagaacaccc ccatcggcga cggccccgtg ctgctgcccg acaaccacta
     7141 cctgagcacc cagtccgccc tgagcaaaga ccccaacgag aagcgcgatc acatggtcct
     7201 gctggagttc gtgaccgccg ccgggatcac tctcggcatg gacgagctgt acaaggaatt
     7261 ctaactagag ctcgctgatc agcctcgact gtgccttcta gttgccagcc atctgttgtt
     7321 tgcccctccc ccgtgccttc cttgaccctg gaaggtgcca ctcccactgt cctttcctaa
     7381 taaaatgagg aaattgcatc gcattgtctg agtaggtgtc attctattct ggggggtggg
     7441 gtggggcagg acagcaaggg ggaggattgg gaagagaata gcaggcatgc tggggagcgg
     7501 ccgcttttgg atcctttacc tgcaggggcg cctgatgcgg tattttctcc ttacgcatct
     7561 gtgcggtatt tcacaccgca tacgtcaaag caaccatagt acgcgccctg tagcggcgca
     7621 ttaagcgcgg cgggtgtggt ggttacgcgc agcgtgaccg ctacacttgc cagcgcccta
     7681 gcgcccgctc ctttcgcttt cttcccttcc tttctcgcca cgttcgccgg ctttccccgt
     7741 caagctctaa atcgggggct ccctttaggg ttccgattta gtgctttacg gcacctcgac
     7801 cccaaaaaac ttgatttggg tgatggttca cgtagtgggc catcgccctg atagacggtt
     7861 tttcgccctt tgacgttgga gtccacgttc tttaatagtg gactcttgtt ccaaactgga
     7921 acaacactca accctatctc gggctattct tttgatttat aagggatttt gccgatttcg
     7981 gcctattggt taaaaaatga gctgatttaa caaaaattta acgcgaattt taacaaaata
     8041 ttaacgttta caattttatg gtgcactctc agtacaatct gctctgatgc cgcatagtta
     8101 agccagcccc gacacccgcc aacacccgct gacgcgccct gacgggcttg tctgctcccg
     8161 gcatccgctt acagacaagc tgtgaccgtc tccgggagct gcatgtgtca gaggttttca
     8221 ccgtcatcac cgaaacgcgc gagacgaaag ggcctcgtga tacgcctatt tttataggtt
     8281 aatgtcatga taataatggt ttcttagacg tcaggtggca cttttcgggg aaatgtgcgc
     8341 ggaaccccta tttgtttatt tttctaaata cattcaaata tgtatccgct catgagacaa
     8401 taaccctgat aaatgcttca ataatattga aaaaggaaga gtatgagtat tcaacatttc
     8461 cgtgtcgccc ttattccctt ttttgcggca ttttgccttc ctgtttttgc tcacccagaa
     8521 acgctggtga aagtaaaaga tgctgaagat cagttgggtg cacgagtggg ttacatcgaa
     8581 ctggatctca acagcggtaa gatccttgag agttttcgcc ccgaagaacg ttttccaatg
     8641 atgagcactt ttaaagttct gctatgtggc gcggtattat cccgtattga cgccgggcaa
     8701 gagcaactcg gtcgccgcat acactattct cagaatgact tggttgagta ctcaccagtc
     8761 acagaaaagc atcttacgga tggcatgaca gtaagagaat tatgcagtgc tgccataacc
     8821 atgagtgata acactgcggc caacttactt ctgacaacga tcggaggacc gaaggagcta
     8881 accgcttttt tgcacaacat gggggatcat gtaactcgcc ttgatcgttg ggaaccggag
     8941 ctgaatgaag ccataccaaa cgacgagcgt gacaccacga tgcctgtagc aatggcaaca
     9001 acgttgcgca aactattaac tggcgaacta cttactctag cttcccggca acaattaata
     9061 gactggatgg aggcggataa agttgcagga ccacttctgc gctcggccct tccggctggc
     9121 tggtttattg ctgataaatc tggagccggt gagcgtggaa gccgcggtat cattgcagca
     9181 ctggggccag atggtaagcc ctcccgtatc gtagttatct acacgacggg gagtcaggca
     9241 actatggatg aacgaaatag acagatcgct gagataggtg cctcactgat taagcattgg
     9301 taactgtcag accaagttta ctcatatata ctttagattg atttaaaact tcatttttaa
     9361 tttaaaagga tctaggtgaa gatccttttt gataatctca tgaccaaaat cccttaacgt
     9421 gagttttcgt tccactgagc gtcagacccc gtagaaaaga tcaaaggatc ttcttgagat
     9481 cctttttttc tgcgcgtaat ctgctgcttg caaacaaaaa aaccaccgct accagcggtg
     9541 gtttgtttgc cggatcaaga gctaccaact ctttttccga aggtaactgg cttcagcaga
     9601 gcgcagatac caaatactgt ccttctagtg tagccgtagt taggccacca cttcaagaac
     9661 tctgtagcac cgcctacata cctcgctctg ctaatcctgt taccagtggc tgctgccagt
     9721 ggcgataagt cgtgtcttac cgggttggac tcaagacgat agttaccgga taaggcgcag
     9781 cggtcgggct gaacgggggg ttcgtgcaca cagcccagct tggagcgaac gacctacacc
     9841 gaactgagat acctacagcg tgagctatga gaaagcgcca cgcttcccga agggagaaag
     9901 gcggacaggt atccggtaag cggcagggtc ggaacaggag agcgcacgag ggagcttcca
     9961 gggggaaacg cctggtatct ttatagtcct gtcgggtttc gccacctctg acttgagcgt
    10021 cgatttttgt gatgctcgtc aggggggcgg agcctatgga aaaacgccag caacgcggcc
    10081 tttttacggt tcctggcctt ttgctggcct tttgctca
//
