LOCUS       sequence-228093-       10125 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 10125)
  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 10125)
  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..10125
                     /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        846..1121
                     /label=chicken beta-actin promoter
     intron          1122..2138
                     /label=chimeric intron
                     /note="chimera between introns from chicken beta-actin and 
                     rabbit beta-globin"
     primer_bind     2062..2084
                     /label=pCAGGS-5
                     /note="Chimeric intron in CAG promoter, forward primer"
     primer_bind     2146..2165
                     /label=pCAG-F
                     /note="Rabbit beta-globin intron, for pCAG plasmids, 
                     forward primer"
     regulatory      2205..2214
                     /label=Kozak sequence
                     /note="vertebrate consensus sequence for strong initiation 
                     of translation (Kozak, 1987)"
                     /regulatory_class="other"
     CDS             2214..2279
                     /codon_start=1
                     /product="three tandem FLAG(R) epitope tags, followed by an
                     enterokinase cleavage site"
                     /label=3xFLAG
                     /translation="DYKDHDGDYKDHDIDYKDDDDK"
     CDS             2286..2306
                     /codon_start=1
                     /product="nuclear localization signal of SV40 (simian virus
                     40) large T antigen"
                     /label=SV40 NLS
                     /translation="PKKKRKV"
     CDS             2331..6431
                     /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             2331..6431
                     /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             6432..6479
                     /codon_start=1
                     /product="bipartite nuclear localization signal from 
                     nucleoplasmin"
                     /label=nucleoplasmin NLS
                     /translation="KRPAATKKAGQAKKKK"
     CDS             6495..6548
                     /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(6549..6551,6552..7262)
                     /codon_start=1
                     /product="enhanced GFP"
                     /label=EGFP
                     /translation="VSKGEELFTGVVPILVELDGDVNGHKFSVSGEGEGDATYGKLTLK
                     FICTTGKLPVPWPTLVTTLTYGVQCFSRYPDHMKQHDFFKSAMPEGYVQERTIFFKDDG
                     NYKTRAEVKFEGDTLVNRIELKGIDFKEDGNILGHKLEYNYNSHNVYIMADKQKNGIKV
                     NFKIRHNIEDGSVQLADHYQQNTPIGDGPVLLPDNHYLSTQSALSKDPNEKRDHMVLLE
                     FVTAAGITLGMDELYK"
     CDS             join(6549..6551,6552..7262)
                     /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(6852..6871)
                     /label=EXFP-R
                     /note="For distinguishing EGFP variants, reverse primer"
     primer_bind     7199..7220
                     /label=EGFP-C
                     /note="EGFP, forward primer"
     primer_bind     complement(7290..7307)
                     /label=BGH-rev
                     /note="Bovine growth hormone terminator, reverse primer. 
                     Also called BGH reverse"
     polyA_signal    7296..7503
                     /label=bGH poly(A) signal
                     /note="bovine growth hormone polyadenylation signal"
     rep_origin      7608..8063
                     /direction=RIGHT
                     /label=f1 ori
                     /note="f1 bacteriophage origin of replication; arrow 
                     indicates direction of (+) strand synthesis"
     primer_bind     complement(7695..7714)
                     /label=F1ori-R
                     /note="F1 origin, reverse primer"
     primer_bind     7905..7926
                     /label=F1ori-F
                     /note="F1 origin, forward primer"
     primer_bind     complement(8080..8099)
                     /label=pRS-marker
                     /note="pRS vectors, use to sequence yeast selectable 
                     marker"
     primer_bind     8199..8221
                     /label=pGEX 3'
                     /note="pGEX vectors, reverse primer"
     primer_bind     complement(8259..8277)
                     /label=pBRforEco
                     /note="pBR322 vectors, upsteam of EcoRI site, forward 
                     primer"
     promoter        8345..8449
                     /gene="bla"
                     /label=AmpR promoter
     CDS             join(8450..8518,8519..9310)
                     /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(8668..8687)
                     /label=Amp-R
                     /note="Ampicillin resistance gene, reverse primer"
     rep_origin      9481..10069
                     /direction=RIGHT
                     /label=ori
                     /note="high-copy-number ColE1/pMB1/pBR322/pUC origin of 
                     replication"
     primer_bind     9970..9989
                     /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 atgggtggag tatttacggt aaactgccca cttggcagta catcaagtgt atcatatgcc
      721 aagtacgccc cctattgacg tcaatgacgg taaatggccc gcctggcatt atgcccagta
      781 catgacctta tgggactttc ctacttggca gtacatctac gtattagtca tcgctattac
      841 catggtcgag gtgagcccca cgttctgctt cactctcccc atctcccccc cctccccacc
      901 cccaattttg tatttattta ttttttaatt attttgtgca gcgatggggg cggggggggg
      961 gggggggcgc gccaggcggg gcggggcggg gcgaggggcg gggcggggcg aggcggagag
     1021 gtgcggcggc agccaatcag agcggcgcgc tccgaaagtt tccttttatg gcgaggcggc
     1081 ggcggcggcg gccctataaa aagcgaagcg cgcggcgggc gggagtcgct gcgcgctgcc
     1141 ttcgccccgt gccccgctcc gccgccgcct cgcgccgccc gccccggctc tgactgaccg
     1201 cgttactccc acaggtgagc gggcgggacg gcccttctcc tccgggctgt aattagcgct
     1261 tggtttaatg acggcttgtt tcttttctgt ggctgcgtga aagccttgag gggctccggg
     1321 agggcccttt gtgcgggggg agcggctcgg ggggtgcgtg cgtgtgtgtg tgcgtgggga
     1381 gcgccgcgtg cggctccgcg ctgcccggcg gctgtgagcg ctgcgggcgc ggcgcggggc
     1441 tttgtgcgct ccgcagtgtg cgcgagggga gcgcggccgg gggcggtgcc ccgcggtgcg
     1501 gggggggctg cgaggggaac aaaggctgcg tgcggggtgt gtgcgtgggg gggtgagcag
     1561 ggggtgtggg cgcgtcggtc gggctgcaac cccccctgca cccccctccc cgagttgctg
     1621 agcacggccc ggcttcgggt gcggggctcc gtacggggcg tggcgcgggg ctcgccgtgc
     1681 cgggcggggg gtggcggcag gtgggggtgc cgggcggggc ggggccgcct cgggccgggg
     1741 agggctcggg ggaggggcgc ggcggccccc ggagcgccgg cggctgtcga ggcgcggcga
     1801 gccgcagcca ttgcctttta tggtaatcgt gcgagagggc gcagggactt cctttgtccc
     1861 aaatctgtgc ggagccgaaa tctgggaggc gccgccgcac cccctctagc gggcgcgggg
     1921 cgaagcggtg cggcgccggc aggaaggaaa tgggcgggga gggccttcgt gcgtcgccgc
     1981 gccgccgtcc ccttctccct ctccagcctc ggggctgtcc gcggggggac ggctgccttc
     2041 gggggggacg gggcagggcg gggttcggct tctggcgtgt gaccggcggc tctagagcct
     2101 ctgctaacca tgttcatgcc ttcttctttt tcctacagct cctgggcaac gtgctggtta
     2161 ttgtgctgtc tcatcatttt ggcaaagaat tcctcgagac cggtgccacc atggactata
     2221 aggaccacga cggagactac aaggatcatg atattgatta caaagacgat gacgataaga
     2281 tggccccaaa gaagaagcgg aaggtcggta tccacggagt cccagcagcc gacaagaagt
     2341 acagcatcgg cctggacatc ggcaccaact ctgtgggctg ggccgtgatc accgacgagt
     2401 acaaggtgcc cagcaagaaa ttcaaggtgc tgggcaacac cgaccggcac agcatcaaga
     2461 agaacctgat cggagccctg ctgttcgaca gcggcgaaac agccgaggcc acccggctga
     2521 agagaaccgc cagaagaaga tacaccagac ggaagaaccg gatctgctat ctgcaagaga
     2581 tcttcagcaa cgagatggcc aaggtggacg acagcttctt ccacagactg gaagagtcct
     2641 tcctggtgga agaggataag aagcacgagc ggcaccccat cttcggcaac atcgtggacg
     2701 aggtggccta ccacgagaag taccccacca tctaccacct gagaaagaaa ctggtggaca
     2761 gcaccgacaa ggccgacctg cggctgatct atctggccct ggcccacatg atcaagttcc
     2821 ggggccactt cctgatcgag ggcgacctga accccgacaa cagcgacgtg gacaagctgt
     2881 tcatccagct ggtgcagacc tacaaccagc tgttcgagga aaaccccatc aacgccagcg
     2941 gcgtggacgc caaggccatc ctgtctgcca gactgagcaa gagcagacgg ctggaaaatc
     3001 tgatcgccca gctgcccggc gagaagaaga atggcctgtt cggaaacctg attgccctga
     3061 gcctgggcct gacccccaac ttcaagagca acttcgacct ggccgaggat gccaaactgc
     3121 agctgagcaa ggacacctac gacgacgacc tggacaacct gctggcccag atcggcgacc
     3181 agtacgccga cctgtttctg gccgccaaga acctgtccga cgccatcctg ctgagcgaca
     3241 tcctgagagt gaacaccgag atcaccaagg cccccctgag cgcctctatg atcaagagat
     3301 acgacgagca ccaccaggac ctgaccctgc tgaaagctct cgtgcggcag cagctgcctg
     3361 agaagtacaa agagattttc ttcgaccaga gcaagaacgg ctacgccggc tacattgacg
     3421 gcggagccag ccaggaagag ttctacaagt tcatcaagcc catcctggaa aagatggacg
     3481 gcaccgagga actgctcgtg aagctgaaca gagaggacct gctgcggaag cagcggacct
     3541 tcgacaacgg cagcatcccc caccagatcc acctgggaga gctgcacgcc attctgcggc
     3601 ggcaggaaga tttttaccca ttcctgaagg acaaccggga aaagatcgag aagatcctga
     3661 ccttccgcat cccctactac gtgggccctc tggccagggg aaacagcaga ttcgcctgga
     3721 tgaccagaaa gagcgaggaa accatcaccc cctggaactt cgaggaagtg gtggacaagg
     3781 gcgcttccgc ccagagcttc atcgagcgga tgaccaactt cgataagaac ctgcccaacg
     3841 agaaggtgct gcccaagcac agcctgctgt acgagtactt caccgtgtat aacgagctga
     3901 ccaaagtgaa atacgtgacc gagggaatga gaaagcccgc cttcctgagc ggcgagcaga
     3961 aaaaggccat cgtggacctg ctgttcaaga ccaaccggaa agtgaccgtg aagcagctga
     4021 aagaggacta cttcaagaaa atcgagtgct tcgactccgt ggaaatctcc ggcgtggaag
     4081 atcggttcaa cgcctccctg ggcacatacc acgatctgct gaaaattatc aaggacaagg
     4141 acttcctgga caatgaggaa aacgaggaca ttctggaaga tatcgtgctg accctgacac
     4201 tgtttgagga cagagagatg atcgaggaac ggctgaaaac ctatgcccac ctgttcgacg
     4261 acaaagtgat gaagcagctg aagcggcgga gatacaccgg ctggggcagg ctgagccgga
     4321 agctgatcaa cggcatccgg gacaagcagt ccggcaagac aatcctggat ttcctgaagt
     4381 ccgacggctt cgccaacaga aacttcatgc agctgatcca cgacgacagc ctgaccttta
     4441 aagaggacat ccagaaagcc caggtgtccg gccagggcga tagcctgcac gagcacattg
     4501 ccaatctggc cggcagcccc gccattaaga agggcatcct gcagacagtg aaggtggtgg
     4561 acgagctcgt gaaagtgatg ggccggcaca agcccgagaa catcgtgatc gaaatggcca
     4621 gagagaacca gaccacccag aagggacaga agaacagccg cgagagaatg aagcggatcg
     4681 aagagggcat caaagagctg ggcagccaga tcctgaaaga acaccccgtg gaaaacaccc
     4741 agctgcagaa cgagaagctg tacctgtact acctgcagaa tgggcgggat atgtacgtgg
     4801 accaggaact ggacatcaac cggctgtccg actacgatgt ggaccatatc gtgcctcaga
     4861 gctttctgaa ggacgactcc atcgacaaca aggtgctgac cagaagcgac aagaaccggg
     4921 gcaagagcga caacgtgccc tccgaagagg tcgtgaagaa gatgaagaac tactggcggc
     4981 agctgctgaa cgccaagctg attacccaga gaaagttcga caatctgacc aaggccgaga
     5041 gaggcggcct gagcgaactg gataaggccg gcttcatcaa gagacagctg gtggaaaccc
     5101 ggcagatcac aaagcacgtg gcacagatcc tggactcccg gatgaacact aagtacgacg
     5161 agaatgacaa gctgatccgg gaagtgaaag tgatcaccct gaagtccaag ctggtgtccg
     5221 atttccggaa ggatttccag ttttacaaag tgcgcgagat caacaactac caccacgccc
     5281 acgacgccta cctgaacgcc gtcgtgggaa ccgccctgat caaaaagtac cctaagctgg
     5341 aaagcgagtt cgtgtacggc gactacaagg tgtacgacgt gcggaagatg atcgccaaga
     5401 gcgagcagga aatcggcaag gctaccgcca agtacttctt ctacagcaac atcatgaact
     5461 ttttcaagac cgagattacc ctggccaacg gcgagatccg gaagcggcct ctgatcgaga
     5521 caaacggcga aaccggggag atcgtgtggg ataagggccg ggattttgcc accgtgcgga
     5581 aagtgctgag catgccccaa gtgaatatcg tgaaaaagac cgaggtgcag acaggcggct
     5641 tcagcaaaga gtctatcctg cccaagagga acagcgataa gctgatcgcc agaaagaagg
     5701 actgggaccc taagaagtac ggcggcttcg acagccccac cgtggcctat tctgtgctgg
     5761 tggtggccaa agtggaaaag ggcaagtcca agaaactgaa gagtgtgaaa gagctgctgg
     5821 ggatcaccat catggaaaga agcagcttcg agaagaatcc catcgacttt ctggaagcca
     5881 agggctacaa agaagtgaaa aaggacctga tcatcaagct gcctaagtac tccctgttcg
     5941 agctggaaaa cggccggaag agaatgctgg cctctgccgg cgaactgcag aagggaaacg
     6001 aactggccct gccctccaaa tatgtgaact tcctgtacct ggccagccac tatgagaagc
     6061 tgaagggctc ccccgaggat aatgagcaga aacagctgtt tgtggaacag cacaagcact
     6121 acctggacga gatcatcgag cagatcagcg agttctccaa gagagtgatc ctggccgacg
     6181 ctaatctgga caaagtgctg tccgcctaca acaagcaccg ggataagccc atcagagagc
     6241 aggccgagaa tatcatccac ctgtttaccc tgaccaatct gggagcccct gccgccttca
     6301 agtactttga caccaccatc gaccggaaga ggtacaccag caccaaagag gtgctggacg
     6361 ccaccctgat ccaccagagc atcaccggcc tgtacgagac acggatcgac ctgtctcagc
     6421 tgggaggcga caaaaggccg gcggccacga aaaaggccgg ccaggcaaaa aagaaaaagg
     6481 aattcggcag tggagagggc agaggaagtc tgctaacatg cggtgacgtc gaggagaatc
     6541 ctggcccagt gagcaagggc gaggagctgt tcaccggggt ggtgcccatc ctggtcgagc
     6601 tggacggcga cgtaaacggc cacaagttca gcgtgtccgg cgagggcgag ggcgatgcca
     6661 cctacggcaa gctgaccctg aagttcatct gcaccaccgg caagctgccc gtgccctggc
     6721 ccaccctcgt gaccaccctg acctacggcg tgcagtgctt cagccgctac cccgaccaca
     6781 tgaagcagca cgacttcttc aagtccgcca tgcccgaagg ctacgtccag gagcgcacca
     6841 tcttcttcaa ggacgacggc aactacaaga cccgcgccga ggtgaagttc gagggcgaca
     6901 ccctggtgaa ccgcatcgag ctgaagggca tcgacttcaa ggaggacggc aacatcctgg
     6961 ggcacaagct ggagtacaac tacaacagcc acaacgtcta tatcatggcc gacaagcaga
     7021 agaacggcat caaggtgaac ttcaagatcc gccacaacat cgaggacggc agcgtgcagc
     7081 tcgccgacca ctaccagcag aacaccccca tcggcgacgg ccccgtgctg ctgcccgaca
     7141 accactacct gagcacccag tccgccctga gcaaagaccc caacgagaag cgcgatcaca
     7201 tggtcctgct ggagttcgtg accgccgccg ggatcactct cggcatggac gagctgtaca
     7261 aggaattcta actagagctc gctgatcagc ctcgactgtg ccttctagtt gccagccatc
     7321 tgttgtttgc ccctcccccg tgccttcctt gaccctggaa ggtgccactc ccactgtcct
     7381 ttcctaataa aatgaggaaa ttgcatcgca ttgtctgagt aggtgtcatt ctattctggg
     7441 gggtggggtg gggcaggaca gcaaggggga ggattgggaa gagaatagca ggcatgctgg
     7501 ggagcggccg cttttggatc ctttacctgc aggggcgcct gatgcggtat tttctcctta
     7561 cgcatctgtg cggtatttca caccgcatac gtcaaagcaa ccatagtacg cgccctgtag
     7621 cggcgcatta agcgcggcgg gtgtggtggt tacgcgcagc gtgaccgcta cacttgccag
     7681 cgccttagcg cccgctcctt tcgctttctt cccttccttt ctcgccacgt tcgccggctt
     7741 tccccgtcaa gctctaaatc gggggctccc tttagggttc cgatttagtg ctttacggca
     7801 cctcgacccc aaaaaacttg atttgggtga tggttcacgt agtgggccat cgccctgata
     7861 gacggttttt cgccctttga cgttggagtc cacgttcttt aatagtggac tcttgttcca
     7921 aactggaaca acactcaact ctatctcggg ctattctttt gatttataag ggattttgcc
     7981 gatttcggtc tattggttaa aaaatgagct gatttaacaa aaatttaacg cgaattttaa
     8041 caaaatatta acgtttacaa ttttatggtg cactctcagt acaatctgct ctgatgccgc
     8101 atagttaagc cagccccgac acccgccaac acccgctgac gcgccctgac gggcttgtct
     8161 gctcccggca tccgcttaca gacaagctgt gaccgtctcc gggagctgca tgtgtcagag
     8221 gttttcaccg tcatcaccga aacgcgcgag acgaaagggc ctcgtgatac gcctattttt
     8281 ataggttaat gtcatgataa taatggtttc ttagacgtca ggtggcactt ttcggggaaa
     8341 tgtgcgcgga acccctattt gtttattttt ctaaatacat tcaaatatgt atccgctcat
     8401 gagacaataa ccctgataaa tgcttcaata atattgaaaa aggaagagta tgagtattca
     8461 acatttccgt gtcgccctta ttcccttttt tgcggcattt tgccttcctg tttttgctca
     8521 cccagaaacg ctggtgaaag taaaagatgc tgaagatcag ttgggtgcac gagtgggtta
     8581 catcgaactg gatctcaaca gcggtaagat ccttgagagt tttcgccccg aagaacgttt
     8641 tccaatgatg agcactttta aagttctgct atgtggcgcg gtattatccc gtattgacgc
     8701 cgggcaagag caactcggtc gccgcataca ctattctcag aatgacttgg ttgagtactc
     8761 accagtcaca gaaaagcatc ttacggatgg catgacagta agagaattat gcagtgctgc
     8821 cataaccatg agtgataaca ctgcggccaa cttacttctg acaacgatcg gaggaccgaa
     8881 ggagctaacc gcttttttgc acaacatggg ggatcatgta actcgccttg atcgttggga
     8941 accggagctg aatgaagcca taccaaacga cgagcgtgac accacgatgc ctgtagcaat
     9001 ggcaacaacg ttgcgcaaac tattaactgg cgaactactt actctagctt cccggcaaca
     9061 attaatagac tggatggagg cggataaagt tgcaggacca cttctgcgct cggcccttcc
     9121 ggctggctgg tttattgctg ataaatctgg agccggtgag cgtggaagcc gcggtatcat
     9181 tgcagcactg gggccagatg gtaagccctc ccgtatcgta gttatctaca cgacggggag
     9241 tcaggcaact atggatgaac gaaatagaca gatcgctgag ataggtgcct cactgattaa
     9301 gcattggtaa ctgtcagacc aagtttactc atatatactt tagattgatt taaaacttca
     9361 tttttaattt aaaaggatct aggtgaagat cctttttgat aatctcatga ccaaaatccc
     9421 ttaacgtgag ttttcgttcc actgagcgtc agaccccgta gaaaagatca aaggatcttc
     9481 ttgagatcct ttttttctgc gcgtaatctg ctgcttgcaa acaaaaaaac caccgctacc
     9541 agcggtggtt tgtttgccgg atcaagagct accaactctt tttccgaagg taactggctt
     9601 cagcagagcg cagataccaa atactgttct tctagtgtag ccgtagttag gccaccactt
     9661 caagaactct gtagcaccgc ctacatacct cgctctgcta atcctgttac cagtggctgc
     9721 tgccagtggc gataagtcgt gtcttaccgg gttggactca agacgatagt taccggataa
     9781 ggcgcagcgg tcgggctgaa cggggggttc gtgcacacag cccagcttgg agcgaacgac
     9841 ctacaccgaa ctgagatacc tacagcgtga gctatgagaa agcgccacgc ttcccgaagg
     9901 gagaaaggcg gacaggtatc cggtaagcgg cagggtcgga acaggagagc gcacgaggga
     9961 gcttccaggg ggaaacgcct ggtatcttta tagtcctgtc gggtttcgcc acctctgact
    10021 tgagcgtcga tttttgtgat gctcgtcagg ggggcggagc ctatggaaaa acgccagcaa
    10081 cgcggccttt ttacggttcc tggccttttg ctggcctttt gctca
//
