LOCUS       sequence-245984-       10417 bp    DNA     circular SYN 15-DEC-2025
DEFINITION  GFP positive gRNA/hSpCas9 construct.
ACCESSION   .
VERSION     .
KEYWORDS    .
SOURCE      synthetic DNA construct
  ORGANISM  synthetic DNA construct
REFERENCE   1  (bases 1 to 10417)
  AUTHORS   Giacalone JC, Sharma TP, Burnight ER, Fingert JF, Mullins RF, Stone 
            EM, Tucker BA
  TITLE     CRISPR-Cas9-Based Genome Editing of Human Induced Pluripotent Stem 
            Cells.
  JOURNAL   Curr Protoc Stem Cell Biol. 2018 Feb 28;44:5B.7.1-5B.7.22. doi: 
            10.1002/cpsc.46.
  PUBMED    29512106
REFERENCE   2  (bases 1 to 10417)
  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: "Curr Protoc
            Stem Cell Biol."; date: "2018-02-28"; volume: "44:5B.7.1-5B.7.22. 
            doi"; pages: " 10.1002/cpsc.46"
COMMENT     Sequence Label: pX330GFP-hU6-gRNA-hSpCas9
FEATURES             Location/Qualifiers
     source          1..10417
                     /mol_type="other DNA"
                     /organism="synthetic DNA construct"
     enhancer        164..543
                     /label=CMV enhancer
                     /note="human cytomegalovirus immediate early enhancer"
     promoter        544..747
                     /label=CMV promoter
                     /note="human cytomegalovirus (CMV) immediate early 
                     promoter"
     primer_bind     698..718
                     /label=CMV-F
                     /note="Human CMV immediate early promoter, forward primer"
     CDS             1006..1671
                     /codon_start=1
                     /product="green fluorescent protein 2 from Pontellina 
                     plumata, also known as ppluGFP2 (Shagin et al., 2004)"
                     /label=CopGFP
                     /translation="MPAMKIECRITGTLNGVEFELVGGGEGTPEQGRMTNKMKSTKGAL
                     TFSPYLLSHVMGYGFYHFGTYPSGYENPFLHAINNGGYTNTRIEKYEDGGVLHVSFSYR
                     YEAGRVIGDFKVVGTGFPEDSVIFTDKIIRSNATVEHLHPMGDNVLVGSFARTFSLRDG
                     GYYSFVVDSHMHFKSAIHPSILQNGGPMFAFRRVEELHSNTELGIVEYQHAFKTPIAFA
                     "
     CDS             1006..1671
                     /codon_start=1
                     /product="green fluorescent protein 2 from Pontellina 
                     plumata, also known as ppluGFP2 (Shagin et al., 2004)"
                     /label=CopGFP
                     /translation="MPAMKIECRITGTLNGVEFELVGGGEGTPEQGRMTNKMKSTKGAL
                     TFSPYLLSHVMGYGFYHFGTYPSGYENPFLHAINNGGYTNTRIEKYEDGGVLHVSFSYR
                     YEAGRVIGDFKVVGTGFPEDSVIFTDKIIRSNATVEHLHPMGDNVLVGSFARTFSLRDG
                     GYYSFVVDSHMHFKSAIHPSILQNGGPMFAFRRVEELHSNTELGIVEYQHAFKTPIAFA
                     "
     primer_bind     complement(1685..1704)
                     /label=EBV-rev
                     /note="SV40 polyA terminator, reverse primer"
     polyA_signal    1714..1795
                     /label=SV40 poly(A) signal
                     /note="SV40 polyadenylation signal"
     primer_bind     1739..1758
                     /label=SV40pA-R
                     /note="SV40 polyA, reverse primer"
     promoter        1938..2186
                     /label=U6 promoter
                     /note="RNA polymerase III promoter for human U6 snRNA 
                     (Domitrovich & Kunkel, 2003)"
     primer_bind     1938..1958
                     /label=hU6-F
                     /note="Human U6 promoter, forward primer"
     primer_bind     2109..2128
                     /label=LKO.1 5'
                     /note="Human U6 promoter, forward primer"
     misc_RNA        2205..2280
                     /label=gRNA scaffold
                     /note="guide RNA scaffold for the Streptococcus pyogenes 
                     CRISPR/Cas9 system"
     enhancer        2377..2662
                     /label=CMV enhancer
                     /note="human cytomegalovirus immediate early enhancer; 
                     contains an 18-bp deletion relative to the standard CMV 
                     enhancer"
     promoter        2664..2941
                     /label=chicken beta-actin promoter
     intron          2942..3170
                     /label=hybrid intron
                     /note="hybrid between chicken beta-actin (CBA) and minute 
                     virus of mice (MMV) introns (Gray et al., 2011)"
     regulatory      3182..3191
                     /label=Kozak sequence
                     /note="vertebrate consensus sequence for strong initiation 
                     of translation (Kozak, 1987)"
                     /regulatory_class="other"
     CDS             3191..3256
                     /codon_start=1
                     /product="three tandem FLAG(R) epitope tags, followed by an
                     enterokinase cleavage site"
                     /label=3xFLAG
                     /translation="DYKDHDGDYKDHDIDYKDDDDK"
     CDS             3263..3283
                     /codon_start=1
                     /product="nuclear localization signal of SV40 (simian virus
                     40) large T antigen"
                     /label=SV40 NLS
                     /translation="PKKKRKV"
     CDS             3308..7408
                     /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             3308..7408
                     /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             7409..7456
                     /codon_start=1
                     /product="bipartite nuclear localization signal from 
                     nucleoplasmin"
                     /label=nucleoplasmin NLS
                     /translation="KRPAATKKAGQAKKKK"
     primer_bind     complement(7484..7501)
                     /label=BGH-rev
                     /note="Bovine growth hormone terminator, reverse primer. 
                     Also called BGH reverse"
     polyA_signal    7490..7697
                     /label=bGH poly(A) signal
                     /note="bovine growth hormone polyadenylation signal"
     repeat_region   7706..7846
                     /label=AAV2 ITR
                     /note="inverted terminal repeat of adeno-associated virus 
                     serotype 2"
     repeat_region   7706..7835
                     /label=AAV2 ITR
     rep_origin      7921..8376
                     /direction=RIGHT
                     /label=f1 ori
                     /note="f1 bacteriophage origin of replication; arrow 
                     indicates direction of (+) strand synthesis"
     primer_bind     complement(8008..8027)
                     /label=F1ori-R
                     /note="F1 origin, reverse primer"
     primer_bind     8218..8239
                     /label=F1ori-F
                     /note="F1 origin, forward primer"
     primer_bind     complement(8393..8412)
                     /label=pRS-marker
                     /note="pRS vectors, use to sequence yeast selectable 
                     marker"
     primer_bind     8512..8534
                     /label=pGEX 3'
                     /note="pGEX vectors, reverse primer"
     primer_bind     complement(8572..8590)
                     /label=pBRforEco
                     /note="pBR322 vectors, upsteam of EcoRI site, forward 
                     primer"
     promoter        8658..8762
                     /gene="bla"
                     /label=AmpR promoter
     CDS             join(8763..8831,8832..9623)
                     /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(8981..9000)
                     /label=Amp-R
                     /note="Ampicillin resistance gene, reverse primer"
     rep_origin      9794..10382
                     /direction=RIGHT
                     /label=ori
                     /note="high-copy-number ColE1/pMB1/pBR322/pUC origin of 
                     replication"
     primer_bind     10283..10302
                     /label=pBR322ori-F
                     /note="pBR322 origin, forward primer"
ORIGIN
        1 cttttgctgg ccttttgctc acatgttcaa tattggccat tagccatatt attcattggt
       61 tatatagcat aaatcaatat tggctattgg ccattgcata cgttgtatct atatcataat
      121 atgtacattt atattggctc atgtccaata tgaccgccat gttggcattg attattgact
      181 agttattaat agtaatcaat tacggggtca ttagttcata gcccatatat ggagttccgc
      241 gttacataac ttacggtaaa tggcccgcct ggctgaccgc ccaacgaccc ccgcccattg
      301 acgtcaataa tgacgtatgt tcccatagta acgccaatag ggactttcca ttgacgtcaa
      361 tgggtggagt atttacggta aactgcccac ttggcagtac atcaagtgta tcatatgcca
      421 agtccgcccc ctattgacgt caatgacggt aaatggcccg cctggcatta tgcccagtac
      481 atgaccttac gggactttcc tacttggcag tacatctacg tattagtcat cgctattacc
      541 atggtgatgc ggttttggca gtacaccaat gggcgtggat agcggtttga ctcacgggga
      601 tttccaagtc tccaccccat tgacgtcaat gggagtttgt tttggcacca aaatcaacgg
      661 gactttccaa aatgtcgtaa taaccccgcc ccgttgacgc aaatgggcgg taggcgtgta
      721 cggtgggagg tctatataag cagrggtcgk ttagkgarcc gtcagatcac tagtagctkt
      781 attgcggtag ttatcacagt taaattgcta acgcagtcag tgctcgactg atcacaggta
      841 agtatcaarr ttacaagaca ggtttaagga ggccaataga aactgggctt gtcgagacag
      901 agaagattct tgcgtttctg ataggcacct attggtctta ctgacatcca ctttgccttt
      961 ctctccacar ggrtaccgaa gccgctagcg ctaccggtcg ccaccatgcc cgccatgaag
     1021 atcgagtgcc gcatcaccgg caccctgaac ggcgtggagt tcgagctggt gggcggcgga
     1081 gagggcaccc ccgagcaggg ccgcatgacc aacaagatga agagcaccaa aggcgccctg
     1141 accttcagcc cctacctgct gagccacgtg atgggctacg gcttctacca cttcggcacc
     1201 taccccagcg gctacgagaa ccccttcctg cacgccatca acaacggcgg ctacaccaac
     1261 acccgcatcg agaagtacga ggacggcggc gtgctgcacg tgagcttcag ctaccgctac
     1321 gaggccggcc gcgtgatcgg cgacttcaag gtggtgggca ccggcttccc cgaggacagc
     1381 gtgatcttca ccgacaagat catccgcagc aacgccaccg tggagcacct gcaccccatg
     1441 ggcgataacg tgctggtggg cagcttcgcc cgcaccttca gcctgcgcga cggcggctac
     1501 tacagcttcg tggtggacag ccacatgcac ttcaagagcg ccatccaccc cagcatcctg
     1561 cagaacgggg gccccatgtt cgccttccgc cgcgtggagg agctgcacag caacaccgag
     1621 ctgggcatcg tggagtacca gcacgccttc aagaccccca tcgccttcgc cagatctcga
     1681 gctcgatgag tttggacaaa ccacaactag aatgcagtga aaaaaatgct ttatttgtga
     1741 aatttgtgat gctattgctt tatttgtaac cattataagc tgcaataaac aagttaacaa
     1801 caacaattgc attcatttta tgtttcaggt tcagggggag gtgtgggagg ttttttaaag
     1861 caagtaaaac ctctacaaat gtggtactta agaggggcag accaaagggc gagacgttaa
     1921 ggcctcacgt gacatgtgag ggcctatttc ccatgattcc ttcatatttg catatacgat
     1981 acaaggctgt tagagagata attggaatta atttgactgt aaacacaaag atattagtac
     2041 aaaatacgtg acgtagaaag taataatttc ttgggtagtt tgcagtttta aaattatgtt
     2101 ttaaaatgga ctatcatatg cttaccgtaa cttgaaagta tttcgatttc ttggctttat
     2161 atatcttgtg gaaaggacga aacaccgggt cttcgagaag acctgtttta gagctagaaa
     2221 tagcaagtta aaataaggct agtccgttat caacttgaaa aagtggcacc gagtcggtgc
     2281 ttttttgttt tagagctaga aatagcaagt taaaataagg ctagtccgtt tttagcgcgt
     2341 gcgccaattc tgcagacaaa tggctctaga ggtacccgtt acataactta cggtaaatgg
     2401 cccgcctggc tgaccgccca acgacccccg cccattgacg tcaatagtaa cgccaatagg
     2461 gactttccat tgacgtcaat gggtggagta tttacggtaa actgcccact tggcagtaca
     2521 tcaagtgtat catatgccaa gtacgccccc tattgacgtc aatgacggta aatggcccgc
     2581 ctggcattgt gcccagtaca tgaccttatg ggactttcct acttggcagt acatctacgt
     2641 attagtcatc gctattacca tggtcgaggt gagccccacg ttctgcttca ctctccccat
     2701 ctcccccccc tccccacccc caattttgta tttatttatt ttttaattat tttgtgcagc
     2761 gatgggggcg gggggggggg gggggcgcgc gccaggcggg gcggggcggg gcgaggggcg
     2821 gggcggggcg aggcggagag gtgcggcggc agccaatcag agcggcgcgc tccgaaagtt
     2881 tccttttatg gcgaggcggc ggcggcggcg gccctataaa aagcgaagcg cgcggcgggc
     2941 gggagtcgct gcgacgctgc cttcgccccg tgccccgctc cgccgccgcc tcgcgccgcc
     3001 cgccccggct ctgactgacc gcgttactcc cacaggtgag cgggcgggac ggcccttctc
     3061 ctccgggctg taattagctg agcaagaggt aagggtttaa gggatggttg gttggtgggg
     3121 tattaatgtt taattacctg gagcacctgc ctgaaatcac tttttttcag gttggaccgg
     3181 tgccaccatg gactataagg accacgacgg agactacaag gatcatgata ttgattacaa
     3241 agacgatgac gataagatgg ccccaaagaa gaagcggaag gtcggtatcc acggagtccc
     3301 agcagccgac aagaagtaca gcatcggcct ggacatcggc accaactctg tgggctgggc
     3361 cgtgatcacc gacgagtaca aggtgcccag caagaaattc aaggtgctgg gcaacaccga
     3421 ccggcacagc atcaagaaga acctgatcgg agccctgctg ttcgacagcg gcgaaacagc
     3481 cgaggccacc cggctgaaga gaaccgccag aagaagatac accagacgga agaaccggat
     3541 ctgctatctg caagagatct tcagcaacga gatggccaag gtggacgaca gcttcttcca
     3601 cagactggaa gagtccttcc tggtggaaga ggataagaag cacgagcggc accccatctt
     3661 cggcaacatc gtggacgagg tggcctacca cgagaagtac cccaccatct accacctgag
     3721 aaagaaactg gtggacagca ccgacaaggc cgacctgcgg ctgatctatc tggccctggc
     3781 ccacatgatc aagttccggg gccacttcct gatcgagggc gacctgaacc ccgacaacag
     3841 cgacgtggac aagctgttca tccagctggt gcagacctac aaccagctgt tcgaggaaaa
     3901 ccccatcaac gccagcggcg tggacgccaa ggccatcctg tctgccagac tgagcaagag
     3961 cagacggctg gaaaatctga tcgcccagct gcccggcgag aagaagaatg gcctgttcgg
     4021 aaacctgatt gccctgagcc tgggcctgac ccccaacttc aagagcaact tcgacctggc
     4081 cgaggatgcc aaactgcagc tgagcaagga cacctacgac gacgacctgg acaacctgct
     4141 ggcccagatc ggcgaccagt acgccgacct gtttctggcc gccaagaacc tgtccgacgc
     4201 catcctgctg agcgacatcc tgagagtgaa caccgagatc accaaggccc ccctgagcgc
     4261 ctctatgatc aagagatacg acgagcacca ccaggacctg accctgctga aagctctcgt
     4321 gcggcagcag ctgcctgaga agtacaaaga gattttcttc gaccagagca agaacggcta
     4381 cgccggctac attgacggcg gagccagcca ggaagagttc tacaagttca tcaagcccat
     4441 cctggaaaag atggacggca ccgaggaact gctcgtgaag ctgaacagag aggacctgct
     4501 gcggaagcag cggaccttcg acaacggcag catcccccac cagatccacc tgggagagct
     4561 gcacgccatt ctgcggcggc aggaagattt ttacccattc ctgaaggaca accgggaaaa
     4621 gatcgagaag atcctgacct tccgcatccc ctactacgtg ggccctctgg ccaggggaaa
     4681 cagcagattc gcctggatga ccagaaagag cgaggaaacc atcaccccct ggaacttcga
     4741 ggaagtggtg gacaagggcg cttccgccca gagcttcatc gagcggatga ccaacttcga
     4801 taagaacctg cccaacgaga aggtgctgcc caagcacagc ctgctgtacg agtacttcac
     4861 cgtgtataac gagctgacca aagtgaaata cgtgaccgag ggaatgagaa agcccgcctt
     4921 cctgagcggc gagcagaaaa aggccatcgt ggacctgctg ttcaagacca accggaaagt
     4981 gaccgtgaag cagctgaaag aggactactt caagaaaatc gagtgcttcg actccgtgga
     5041 aatctccggc gtggaagatc ggttcaacgc ctccctgggc acataccacg atctgctgaa
     5101 aattatcaag gacaaggact tcctggacaa tgaggaaaac gaggacattc tggaagatat
     5161 cgtgctgacc ctgacactgt ttgaggacag agagatgatc gaggaacggc tgaaaaccta
     5221 tgcccacctg ttcgacgaca aagtgatgaa gcagctgaag cggcggagat acaccggctg
     5281 gggcaggctg agccggaagc tgatcaacgg catccgggac aagcagtccg gcaagacaat
     5341 cctggatttc ctgaagtccg acggcttcgc caacagaaac ttcatgcagc tgatccacga
     5401 cgacagcctg acctttaaag aggacatcca gaaagcccag gtgtccggcc agggcgatag
     5461 cctgcacgag cacattgcca atctggccgg cagccccgcc attaagaagg gcatcctgca
     5521 gacagtgaag gtggtggacg agctcgtgaa agtgatgggc cggcacaagc ccgagaacat
     5581 cgtgatcgaa atggccagag agaaccagac cacccagaag ggacagaaga acagccgcga
     5641 gagaatgaag cggatcgaag agggcatcaa agagctgggc agccagatcc tgaaagaaca
     5701 ccccgtggaa aacacccagc tgcagaacga gaagctgtac ctgtactacc tgcagaatgg
     5761 gcgggatatg tacgtggacc aggaactgga catcaaccgg ctgtccgact acgatgtgga
     5821 ccatatcgtg cctcagagct ttctgaagga cgactccatc gacaacaagg tgctgaccag
     5881 aagcgacaag aaccggggca agagcgacaa cgtgccctcc gaagaggtcg tgaagaagat
     5941 gaagaactac tggcggcagc tgctgaacgc caagctgatt acccagagaa agttcgacaa
     6001 tctgaccaag gccgagagag gcggcctgag cgaactggat aaggccggct tcatcaagag
     6061 acagctggtg gaaacccggc agatcacaaa gcacgtggca cagatcctgg actcccggat
     6121 gaacactaag tacgacgaga atgacaagct gatccgggaa gtgaaagtga tcaccctgaa
     6181 gtccaagctg gtgtccgatt tccggaagga tttccagttt tacaaagtgc gcgagatcaa
     6241 caactaccac cacgcccacg acgcctacct gaacgccgtc gtgggaaccg ccctgatcaa
     6301 aaagtaccct aagctggaaa gcgagttcgt gtacggcgac tacaaggtgt acgacgtgcg
     6361 gaagatgatc gccaagagcg agcaggaaat cggcaaggct accgccaagt acttcttcta
     6421 cagcaacatc atgaactttt tcaagaccga gattaccctg gccaacggcg agatccggaa
     6481 gcggcctctg atcgagacaa acggcgaaac cggggagatc gtgtgggata agggccggga
     6541 ttttgccacc gtgcggaaag tgctgagcat gccccaagtg aatatcgtga aaaagaccga
     6601 ggtgcagaca ggcggcttca gcaaagagtc tatcctgccc aagaggaaca gcgataagct
     6661 gatcgccaga aagaaggact gggaccctaa gaagtacggc ggcttcgaca gccccaccgt
     6721 ggcctattct gtgctggtgg tggccaaagt ggaaaagggc aagtccaaga aactgaagag
     6781 tgtgaaagag ctgctgggga tcaccatcat ggaaagaagc agcttcgaga agaatcccat
     6841 cgactttctg gaagccaagg gctacaaaga agtgaaaaag gacctgatca tcaagctgcc
     6901 taagtactcc ctgttcgagc tggaaaacgg ccggaagaga atgctggcct ctgccggcga
     6961 actgcagaag ggaaacgaac tggccctgcc ctccaaatat gtgaacttcc tgtacctggc
     7021 cagccactat gagaagctga agggctcccc cgaggataat gagcagaaac agctgtttgt
     7081 ggaacagcac aagcactacc tggacgagat catcgagcag atcagcgagt tctccaagag
     7141 agtgatcctg gccgacgcta atctggacaa agtgctgtcc gcctacaaca agcaccggga
     7201 taagcccatc agagagcagg ccgagaatat catccacctg tttaccctga ccaatctggg
     7261 agcccctgcc gccttcaagt actttgacac caccatcgac cggaagaggt acaccagcac
     7321 caaagaggtg ctggacgcca ccctgatcca ccagagcatc accggcctgt acgagacacg
     7381 gatcgacctg tctcagctgg gaggcgacaa aaggccggcg gccacgaaaa aggccggcca
     7441 ggcaaaaaag aaaaagtaag aattcctaga gctcgctgat cagcctcgac tgtgccttct
     7501 agttgccagc catctgttgt ttgcccctcc cccgtgcctt ccttgaccct ggaaggtgcc
     7561 actcccactg tcctttccta ataaaatgag gaaattgcat cgcattgtct gagtaggtgt
     7621 cattctattc tggggggtgg ggtggggcag gacagcaagg gggaggattg ggaagagaat
     7681 agcaggcatg ctggggagcg gccgcaggaa cccctagtga tggagttggc cactccctct
     7741 ctgcgcgctc gctcgctcac tgaggccggg cgaccaaagg tcgcccgacg cccgggcttt
     7801 gcccgggcgg cctcagtgag cgagcgagcg cgcagctgcc tgcaggggcg cctgatgcgg
     7861 tattttctcc ttacgcatct gtgcggtatt tcacaccgca tacgtcaaag caaccatagt
     7921 acgcgccctg tagcggcgca ttaagcgcgg cgggtgtggt ggttacgcgc agcgtgaccg
     7981 ctacacttgc cagcgcccta gcgcccgctc ctttcgcttt cttcccttcc tttctcgcca
     8041 cgttcgccgg ctttccccgt caagctctaa atcgggggct ccctttaggg ttccgattta
     8101 gtgctttacg gcacctcgac cccaaaaaac ttgatttggg tgatggttca cgtagtgggc
     8161 catcgccctg atagacggtt tttcgccctt tgacgttgga gtccacgttc tttaatagtg
     8221 gactcttgtt ccaaactgga acaacactca accctatctc gggctattct tttgatttat
     8281 aagggatttt gccgatttcg gcctattggt taaaaaatga gctgatttaa caaaaattta
     8341 acgcgaattt taacaaaata ttaacgttta caattttatg gtgcactctc agtacaatct
     8401 gctctgatgc cgcatagtta agccagcccc gacacccgcc aacacccgct gacgcgccct
     8461 gacgggcttg tctgctcccg gcatccgctt acagacaagc tgtgaccgtc tccgggagct
     8521 gcatgtgtca gaggttttca ccgtcatcac cgaaacgcgc gagacgaaag ggcctcgtga
     8581 tacgcctatt tttataggtt aatgtcatga taataatggt ttcttagacg tcaggtggca
     8641 cttttcgggg aaatgtgcgc ggaaccccta tttgtttatt tttctaaata cattcaaata
     8701 tgtatccgct catgagacaa taaccctgat aaatgcttca ataatattga aaaaggaaga
     8761 gtatgagtat tcaacatttc cgtgtcgccc ttattccctt ttttgcggca ttttgccttc
     8821 ctgtttttgc tcacccagaa acgctggtga aagtaaaaga tgctgaagat cagttgggtg
     8881 cacgagtggg ttacatcgaa ctggatctca acagcggtaa gatccttgag agttttcgcc
     8941 ccgaagaacg ttttccaatg atgagcactt ttaaagttct gctatgtggc gcggtattat
     9001 cccgtattga cgccgggcaa gagcaactcg gtcgccgcat acactattct cagaatgact
     9061 tggttgagta ctcaccagtc acagaaaagc atcttacgga tggcatgaca gtaagagaat
     9121 tatgcagtgc tgccataacc atgagtgata acactgcggc caacttactt ctgacaacga
     9181 tcggaggacc gaaggagcta accgcttttt tgcacaacat gggggatcat gtaactcgcc
     9241 ttgatcgttg ggaaccggag ctgaatgaag ccataccaaa cgacgagcgt gacaccacga
     9301 tgcctgtagc aatggcaaca acgttgcgca aactattaac tggcgaacta cttactctag
     9361 cttcccggca acaattaata gactggatgg aggcggataa agttgcagga ccacttctgc
     9421 gctcggccct tccggctggc tggtttattg ctgataaatc tggagccggt gagcgtggaa
     9481 gccgcggtat cattgcagca ctggggccag atggtaagcc ctcccgtatc gtagttatct
     9541 acacgacggg gagtcaggca actatggatg aacgaaatag acagatcgct gagataggtg
     9601 cctcactgat taagcattgg taactgtcag accaagttta ctcatatata ctttagattg
     9661 atttaaaact tcatttttaa tttaaaagga tctaggtgaa gatccttttt gataatctca
     9721 tgaccaaaat cccttaacgt gagttttcgt tccactgagc gtcagacccc gtagaaaaga
     9781 tcaaaggatc ttcttgagat cctttttttc tgcgcgtaat ctgctgcttg caaacaaaaa
     9841 aaccaccgct accagcggtg gtttgtttgc cggatcaaga gctaccaact ctttttccga
     9901 aggtaactgg cttcagcaga gcgcagatac caaatactgt ccttctagtg tagccgtagt
     9961 taggccacca cttcaagaac tctgtagcac cgcctacata cctcgctctg ctaatcctgt
    10021 taccagtggc tgctgccagt ggcgataagt cgtgtcttac cgggttggac tcaagacgat
    10081 agttaccgga taaggcgcag cggtcgggct gaacgggggg ttcgtgcaca cagcccagct
    10141 tggagcgaac gacctacacc gaactgagat acctacagcg tgagctatga gaaagcgcca
    10201 cgcttcccga agggagaaag gcggacaggt atccggtaag cggcagggtc ggaacaggag
    10261 agcgcacgag ggagcttcca gggggaaacg cctggtatct ttatagtcct gtcgggtttc
    10321 gccacctctg acttgagcgt cgatttttgt gatgctcgtc aggggggcgg agcctatgga
    10381 aaaacgccag caacgcggcc tttttacggt tcctggc
//
