LOCUS       sequence-343191-        9691 bp    DNA     circular SYN 15-DEC-2025
DEFINITION  Cas9-2A-GFP expression vector bearing two sgRNAs targeting the 
            centromeric human SOX17 CTCF-boundary..
ACCESSION   .
VERSION     .
KEYWORDS    .
SOURCE      synthetic DNA construct
  ORGANISM  synthetic DNA construct
REFERENCE   1  (bases 1 to 9691)
  AUTHORS   Wu HJ, Landshammer A, Stamenova EK, Bolondi A, Kretzmer H, Meissner 
            A, Michor F
  TITLE     Topological isolation of developmental regulators in mammalian 
            genomes.
  JOURNAL   Nat Commun. 2021 Aug 12;12(1):4897. doi: 10.1038/s41467-021-24951-7.
  PUBMED    34385432
REFERENCE   2  (bases 1 to 9691)
  AUTHORS   .
  TITLE     Direct Submission
  JOURNAL   Exported Dec 15, 2025 from SnapGene Server 8.0.1
            https://www.snapgene.com
COMMENT     SGRef: number: 1; type: "Journal Article"; journalName: "Nat 
            Commun."; date: "2021-08-12"; volume: "12(1):4897. doi"; pages: " 
            10.1038/s41467-021-24951-7"
COMMENT     Sequence Label: 2X_pX458_pSpCas9(BB)-2A-GFP_SOX17_bKO
FEATURES             Location/Qualifiers
     source          1..9691
                     /mol_type="other DNA"
                     /organism="synthetic DNA construct"
     promoter        25..273
                     /label=U6 promoter
                     /note="RNA polymerase III promoter for human U6 snRNA 
                     (Domitrovich & Kunkel, 2003)"
     primer_bind     25..45
                     /label=hU6-F
                     /note="Human U6 promoter, forward primer"
     misc_RNA        294..369
                     /label=gRNA scaffold
                     /note="guide RNA scaffold for the Streptococcus pyogenes 
                     CRISPR/Cas9 system"
     misc_RNA        735..810
                     /label=gRNA scaffold
                     /note="guide RNA scaffold for the Streptococcus pyogenes 
                     CRISPR/Cas9 system"
     enhancer        879..1164
                     /label=CMV enhancer
                     /note="human cytomegalovirus immediate early enhancer; 
                     contains an 18-bp deletion relative to the standard CMV 
                     enhancer"
     intron          1432..1659
                     /label=hybrid intron
                     /note="hybrid between chicken beta-actin (CBA) and minute 
                     virus of mice (MMV) introns (Gray et al., 2011)"
     regulatory      1671..1680
                     /label=Kozak sequence
                     /note="vertebrate consensus sequence for strong initiation 
                     of translation (Kozak, 1987)"
                     /regulatory_class="other"
     CDS             1680..1745
                     /codon_start=1
                     /product="three tandem FLAG(R) epitope tags, followed by an
                     enterokinase cleavage site"
                     /label=3xFLAG
                     /translation="DYKDHDGDYKDHDIDYKDDDDK"
     CDS             1752..1772
                     /codon_start=1
                     /product="nuclear localization signal of SV40 (simian virus
                     40) large T antigen"
                     /label=SV40 NLS
                     /translation="PKKKRKV"
     CDS             1797..5897
                     /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             1797..5897
                     /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             5898..5945
                     /codon_start=1
                     /product="bipartite nuclear localization signal from 
                     nucleoplasmin"
                     /label=nucleoplasmin NLS
                     /translation="KRPAATKKAGQAKKKK"
     CDS             5961..6014
                     /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(6015..6017,6018..6728)
                     /codon_start=1
                     /product="enhanced GFP"
                     /label=EGFP
                     /translation="VSKGEELFTGVVPILVELDGDVNGHKFSVSGEGEGDATYGKLTLK
                     FICTTGKLPVPWPTLVTTLTYGVQCFSRYPDHMKQHDFFKSAMPEGYVQERTIFFKDDG
                     NYKTRAEVKFEGDTLVNRIELKGIDFKEDGNILGHKLEYNYNSHNVYIMADKQKNGIKV
                     NFKIRHNIEDGSVQLADHYQQNTPIGDGPVLLPDNHYLSTQSALSKDPNEKRDHMVLLE
                     FVTAAGITLGMDELYK"
     CDS             join(6015..6017,6018..6728)
                     /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(6318..6337)
                     /label=EXFP-R
                     /note="For distinguishing EGFP variants, reverse primer"
     primer_bind     6665..6686
                     /label=EGFP-C
                     /note="EGFP, forward primer"
     primer_bind     complement(6756..6773)
                     /label=BGH-rev
                     /note="Bovine growth hormone terminator, reverse primer. 
                     Also called BGH reverse"
     polyA_signal    6762..6969
                     /label=bGH poly(A) signal
                     /note="bovine growth hormone polyadenylation signal"
     repeat_region   6978..7118
                     /label=AAV2 ITR
                     /note="inverted terminal repeat of adeno-associated virus 
                     serotype 2"
     repeat_region   6978..7107
                     /label=AAV2 ITR
     rep_origin      7193..7648
                     /direction=RIGHT
                     /label=f1 ori
                     /note="f1 bacteriophage origin of replication; arrow 
                     indicates direction of (+) strand synthesis"
     primer_bind     complement(7280..7299)
                     /label=F1ori-R
                     /note="F1 origin, reverse primer"
     primer_bind     7490..7511
                     /label=F1ori-F
                     /note="F1 origin, forward primer"
     primer_bind     complement(7665..7684)
                     /label=pRS-marker
                     /note="pRS vectors, use to sequence yeast selectable 
                     marker"
     primer_bind     7784..7806
                     /label=pGEX 3'
                     /note="pGEX vectors, reverse primer"
     primer_bind     complement(7844..7862)
                     /label=pBRforEco
                     /note="pBR322 vectors, upsteam of EcoRI site, forward 
                     primer"
     promoter        7930..8034
                     /gene="bla"
                     /label=AmpR promoter
     CDS             join(8035..8103,8104..8895)
                     /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(8253..8272)
                     /label=Amp-R
                     /note="Ampicillin resistance gene, reverse primer"
     rep_origin      9066..9654
                     /direction=RIGHT
                     /label=ori
                     /note="high-copy-number ColE1/pMB1/pBR322/pUC origin of 
                     replication"
     primer_bind     9555..9574
                     /label=pBR322ori-F
                     /note="pBR322 origin, forward primer"
ORIGIN
        1 tttgctggcc ttttgctcac atgtgagggc ctatttccca tgattccttc atatttgcat
       61 atacgataca aggctgttag agagataatt ggaattaatt tgactgtaaa cacaaagata
      121 ttagtacaaa atacgtgacg tagaaagtaa taatttcttg ggtagtttgc agttttaaaa
      181 ttatgtttta aaatggacta tcatatgctt accgtaactt gaaagtattt cgatttcttg
      241 gctttatata tcttgtggaa aggacgaaac accgggctgc accaaatcgc cacgttttag
      301 agctagaaat agcaagttaa aataaggcta gtccgttatc aacttgaaaa agtggcaccg
      361 agtcggtgct tttttgtttt agagctagaa atagcaagtt aaaataaggc tagtccgttt
      421 ttagcgcgtg cgccaattct gcagacaaat ggctctagag gtaccaatat gcattttccc
      481 atgattcctt catatttgca tatacgatac aaggctgtta gagagataat tggaattaat
      541 ttgactgtaa acacaaagat attagtacaa aatacgtgac gtagaaagta ataatttctt
      601 gggtagtttg cagttttaaa attatgtttt aaaatggact atcatatgct taccgtaact
      661 tgaaagtatt tcgatttctt ggctttatat atcttgtgga aaggacgaaa caccacatcc
      721 agtctgccaa catagtttta gagctagaaa tagcaagtta aaataaggct agtccgttat
      781 caacttgaaa aagtggcacc gagtcggtgc ttttttgttt tcctgcagga gatttagcgc
      841 gtgcgccaat tctgcagaca aatggctcta gaggtacccg ttacataact tacggtaaat
      901 ggcccgcctg gctgaccgcc caacgacccc cgcccattga cgtcaatagt aacgccaata
      961 gggactttcc attgacgtca atgggtggag tatttacggt aaactgccca cttggcagta
     1021 catcaagtgt atcatatgcc aagtacgccc cctattgacg tcaatgacgg taaatggccc
     1081 gcctggcatt gtgcccagta catgacctta tgggactttc ctacttggca gtacatctac
     1141 gtattagtca tcgctattac catggtcgag gtgagcccca cgttctgctt cactctcccc
     1201 atctcccccc cctccccacc cccaattttg tatttattta ttttttaatt attttgtgca
     1261 gcgatggggg cggggggggg gggggggggg gcggggcggg gcgaggggcg gggcggggcg
     1321 aggcggagag gtgcggcggc agccaatcag agcggcgcgc tccgaaagtt tccttttatg
     1381 gcgaggcggc ggcggcggcg gccctataaa aagcgaagcg cgcggcgggc gggagtcgct
     1441 gcgcgctgcc ttcgccccgt gccccgctcc gccgccgcct cgcgccgccc gccccggctc
     1501 tgactgaccg cgttactccc acaggtgagc gggcgggacg gcccttctcc tccgggctgt
     1561 aattagctga gcaagaggta agggtttaag ggatggttgg ttggtggggt attaatgttt
     1621 aattacctgg agcacctgcc tgaaatcact ttttttcagg ttggaccggt gccaccatgg
     1681 actataagga ccacgacgga gactacaagg atcatgatat tgattacaaa gacgatgacg
     1741 ataagatggc cccaaagaag aagcggaagg tcggtatcca cggagtccca gcagccgaca
     1801 agaagtacag catcggcctg gacatcggca ccaactctgt gggctgggcc gtgatcaccg
     1861 acgagtacaa ggtgcccagc aagaaattca aggtgctggg caacaccgac cggcacagca
     1921 tcaagaagaa cctgatcgga gccctgctgt tcgacagcgg cgaaacagcc gaggccaccc
     1981 ggctgaagag aaccgccaga agaagataca ccagacggaa gaaccggatc tgctatctgc
     2041 aagagatctt cagcaacgag atggccaagg tggacgacag cttcttccac agactggaag
     2101 agtccttcct ggtggaagag gataagaagc acgagcggca ccccatcttc ggcaacatcg
     2161 tggacgaggt ggcctaccac gagaagtacc ccaccatcta ccacctgaga aagaaactgg
     2221 tggacagcac cgacaaggcc gacctgcggc tgatctatct ggccctggcc cacatgatca
     2281 agttccgggg ccacttcctg atcgagggcg acctgaaccc cgacaacagc gacgtggaca
     2341 agctgttcat ccagctggtg cagacctaca accagctgtt cgaggaaaac cccatcaacg
     2401 ccagcggcgt ggacgccaag gccatcctgt ctgccagact gagcaagagc agacggctgg
     2461 aaaatctgat cgcccagctg cccggcgaga agaagaatgg cctgttcgga aacctgattg
     2521 ccctgagcct gggcctgacc cccaacttca agagcaactt cgacctggcc gaggatgcca
     2581 aactgcagct gagcaaggac acctacgacg acgacctgga caacctgctg gcccagatcg
     2641 gcgaccagta cgccgacctg tttctggccg ccaagaacct gtccgacgcc atcctgctga
     2701 gcgacatcct gagagtgaac accgagatca ccaaggcccc cctgagcgcc tctatgatca
     2761 agagatacga cgagcaccac caggacctga ccctgctgaa agctctcgtg cggcagcagc
     2821 tgcctgagaa gtacaaagag attttcttcg accagagcaa gaacggctac gccggctaca
     2881 ttgacggcgg agccagccag gaagagttct acaagttcat caagcccatc ctggaaaaga
     2941 tggacggcac cgaggaactg ctcgtgaagc tgaacagaga ggacctgctg cggaagcagc
     3001 ggaccttcga caacggcagc atcccccacc agatccacct gggagagctg cacgccattc
     3061 tgcggcggca ggaagatttt tacccattcc tgaaggacaa ccgggaaaag atcgagaaga
     3121 tcctgacctt ccgcatcccc tactacgtgg gccctctggc caggggaaac agcagattcg
     3181 cctggatgac cagaaagagc gaggaaacca tcaccccctg gaacttcgag gaagtggtgg
     3241 acaagggcgc ttccgcccag agcttcatcg agcggatgac caacttcgat aagaacctgc
     3301 ccaacgagaa ggtgctgccc aagcacagcc tgctgtacga gtacttcacc gtgtataacg
     3361 agctgaccaa agtgaaatac gtgaccgagg gaatgagaaa gcccgccttc ctgagcggcg
     3421 agcagaaaaa ggccatcgtg gacctgctgt tcaagaccaa ccggaaagtg accgtgaagc
     3481 agctgaaaga ggactacttc aagaaaatcg agtgcttcga ctccgtggaa atctccggcg
     3541 tggaagatcg gttcaacgcc tccctgggca cataccacga tctgctgaaa attatcaagg
     3601 acaaggactt cctggacaat gaggaaaacg aggacattct ggaagatatc gtgctgaccc
     3661 tgacactgtt tgaggacaga gagatgatcg aggaacggct gaaaacctat gcccacctgt
     3721 tcgacgacaa agtgatgaag cagctgaagc ggcggagata caccggctgg ggcaggctga
     3781 gccggaagct gatcaacggc atccgggaca agcagtccgg caagacaatc ctggatttcc
     3841 tgaagtccga cggcttcgcc aacagaaact tcatgcagct gatccacgac gacagcctga
     3901 cctttaaaga ggacatccag aaagcccagg tgtccggcca gggcgatagc ctgcacgagc
     3961 acattgccaa tctggccggc agccccgcca ttaagaaggg catcctgcag acagtgaagg
     4021 tggtggacga gctcgtgaaa gtgatgggcc ggcacaagcc cgagaacatc gtgatcgaaa
     4081 tggccagaga gaaccagacc acccagaagg gacagaagaa cagccgcgag agaatgaagc
     4141 ggatcgaaga gggcatcaaa gagctgggca gccagatcct gaaagaacac cccgtggaaa
     4201 acacccagct gcagaacgag aagctgtacc tgtactacct gcagaatggg cgggatatgt
     4261 acgtggacca ggaactggac atcaaccggc tgtccgacta cgatgtggac catatcgtgc
     4321 ctcagagctt tctgaaggac gactccatcg acaacaaggt gctgaccaga agcgacaaga
     4381 accggggcaa gagcgacaac gtgccctccg aagaggtcgt gaagaagatg aagaactact
     4441 ggcggcagct gctgaacgcc aagctgatta cccagagaaa gttcgacaat ctgaccaagg
     4501 ccgagagagg cggcctgagc gaactggata aggccggctt catcaagaga cagctggtgg
     4561 aaacccggca gatcacaaag cacgtggcac agatcctgga ctcccggatg aacactaagt
     4621 acgacgagaa tgacaagctg atccgggaag tgaaagtgat caccctgaag tccaagctgg
     4681 tgtccgattt ccggaaggat ttccagtttt acaaagtgcg cgagatcaac aactaccacc
     4741 acgcccacga cgcctacctg aacgccgtcg tgggaaccgc cctgatcaaa aagtacccta
     4801 agctggaaag cgagttcgtg tacggcgact acaaggtgta cgacgtgcgg aagatgatcg
     4861 ccaagagcga gcaggaaatc ggcaaggcta ccgccaagta cttcttctac agcaacatca
     4921 tgaacttttt caagaccgag attaccctgg ccaacggcga gatccggaag cggcctctga
     4981 tcgagacaaa cggcgaaacc ggggagatcg tgtgggataa gggccgggat tttgccaccg
     5041 tgcggaaagt gctgagcatg ccccaagtga atatcgtgaa aaagaccgag gtgcagacag
     5101 gcggcttcag caaagagtct atcctgccca agaggaacag cgataagctg atcgccagaa
     5161 agaaggactg ggaccctaag aagtacggcg gcttcgacag ccccaccgtg gcctattctg
     5221 tgctggtggt ggccaaagtg gaaaagggca agtccaagaa actgaagagt gtgaaagagc
     5281 tgctggggat caccatcatg gaaagaagca gcttcgagaa gaatcccatc gactttctgg
     5341 aagccaaggg ctacaaagaa gtgaaaaagg acctgatcat caagctgcct aagtactccc
     5401 tgttcgagct ggaaaacggc cggaagagaa tgctggcctc tgccggcgaa ctgcagaagg
     5461 gaaacgaact ggccctgccc tccaaatatg tgaacttcct gtacctggcc agccactatg
     5521 agaagctgaa gggctccccc gaggataatg agcagaaaca gctgtttgtg gaacagcaca
     5581 agcactacct ggacgagatc atcgagcaga tcagcgagtt ctccaagaga gtgatcctgg
     5641 ccgacgctaa tctggacaaa gtgctgtccg cctacaacaa gcaccgggat aagcccatca
     5701 gagagcaggc cgagaatatc atccacctgt ttaccctgac caatctggga gcccctgccg
     5761 ccttcaagta ctttgacacc accatcgacc ggaagaggta caccagcacc aaagaggtgc
     5821 tggacgccac cctgatccac cagagcatca ccggcctgta cgagacacgg atcgacctgt
     5881 ctcagctggg aggcgacaaa aggccggcgg ccacgaaaaa ggccggccag gcaaaaaaga
     5941 aaaaggaatt cggcagtgga gagggcagag gaagtctgct aacatgcggt gacgtcgagg
     6001 agaatcctgg cccagtgagc aagggcgagg agctgttcac cggggtggtg cccatcctgg
     6061 tcgagctgga cggcgacgta aacggccaca agttcagcgt gtccggcgag ggcgagggcg
     6121 atgccaccta cggcaagctg accctgaagt tcatctgcac caccggcaag ctgcccgtgc
     6181 cctggcccac cctcgtgacc accctgacct acggcgtgca gtgcttcagc cgctaccccg
     6241 accacatgaa gcagcacgac ttcttcaagt ccgccatgcc cgaaggctac gtccaggagc
     6301 gcaccatctt cttcaaggac gacggcaact acaagacccg cgccgaggtg aagttcgagg
     6361 gcgacaccct ggtgaaccgc atcgagctga agggcatcga cttcaaggag gacggcaaca
     6421 tcctggggca caagctggag tacaactaca acagccacaa cgtctatatc atggccgaca
     6481 agcagaagaa cggcatcaag gtgaacttca agatccgcca caacatcgag gacggcagcg
     6541 tgcagctcgc cgaccactac cagcagaaca cccccatcgg cgacggcccc gtgctgctgc
     6601 ccgacaacca ctacctgagc acccagtccg ccctgagcaa agaccccaac gagaagcgcg
     6661 atcacatggt cctgctggag ttcgtgaccg ccgccgggat cactctcggc atggacgagc
     6721 tgtacaagga attctaacta gagctcgctg atcagcctcg actgtgcctt ctagttgcca
     6781 gccatctgtt gtttgcccct cccccgtgcc ttccttgacc ctggaaggtg ccactcccac
     6841 tgtcctttcc taataaaatg aggaaattgc atcgcattgt ctgagtaggt gtcattctat
     6901 tctggggggt ggggtggggc aggacagcaa gggggaggat tgggaagaga atagcaggca
     6961 tgctggggag cggccgcagg aacccctagt gatggagttg gccactccct ctctgcgcgc
     7021 tcgctcgctc actgaggccg ggcgaccaaa ggtcgcccga cgcccgggct ttgcccgggc
     7081 ggcctcagtg agcgagcgag cgcgcagctg cctgcagggg cgcctgatgc ggtattttct
     7141 ccttacgcat ctgtgcggta tttcacaccg catacgtcaa agcaaccata gtacgcgccc
     7201 tgtagcggcg cattaagcgc ggcgggtgtg gtggttacgc gcagcgtgac cgctacactt
     7261 gccagcgcct tagcgcccgc tcctttcgct ttcttccctt cctttctcgc cacgttcgcc
     7321 ggctttcccc gtcaagctct aaatcggggg ctccctttag ggttccgatt tagtgcttta
     7381 cggcacctcg accccaaaaa acttgatttg ggtgatggtt cacgtagtgg gccatcgccc
     7441 tgatagacgg tttttcgccc tttgacgttg gagtccacgt tctttaatag tggactcttg
     7501 ttccaaactg gaacaacact caactctatc tcgggctatt cttttgattt ataagggatt
     7561 ttgccgattt cggtctattg gttaaaaaat gagctgattt aacaaaaatt taacgcgaat
     7621 tttaacaaaa tattaacgtt tacaatttta tggtgcactc tcagtacaat ctgctctgat
     7681 gccgcatagt taagccagcc ccgacacccg ccaacacccg ctgacgcgcc ctgacgggct
     7741 tgtctgctcc cggcatccgc ttacagacaa gctgtgaccg tctccgggag ctgcatgtgt
     7801 cagaggtttt caccgtcatc accgaaacgc gcgagacgaa agggcctcgt gatacgccta
     7861 tttttatagg ttaatgtcat gataataatg gtttcttaga cgtcaggtgg cacttttcgg
     7921 ggaaatgtgc gcggaacccc tatttgttta tttttctaaa tacattcaaa tatgtatccg
     7981 ctcatgagac aataaccctg ataaatgctt caataatatt gaaaaaggaa gagtatgagt
     8041 attcaacatt tccgtgtcgc ccttattccc ttttttgcgg cattttgcct tcctgttttt
     8101 gctcacccag aaacgctggt gaaagtaaaa gatgctgaag atcagttggg tgcacgagtg
     8161 ggttacatcg aactggatct caacagcggt aagatccttg agagttttcg ccccgaagaa
     8221 cgttttccaa tgatgagcac ttttaaagtt ctgctatgtg gcgcggtatt atcccgtatt
     8281 gacgccgggc aagagcaact cggtcgccgc atacactatt ctcagaatga cttggttgag
     8341 tactcaccag tcacagaaaa gcatcttacg gatggcatga cagtaagaga attatgcagt
     8401 gctgccataa ccatgagtga taacactgcg gccaacttac ttctgacaac gatcggagga
     8461 ccgaaggagc taaccgcttt tttgcacaac atgggggatc atgtaactcg ccttgatcgt
     8521 tgggaaccgg agctgaatga agccatacca aacgacgagc gtgacaccac gatgcctgta
     8581 gcaatggcaa caacgttgcg caaactatta actggcgaac tacttactct agcttcccgg
     8641 caacaattaa tagactggat ggaggcggat aaagttgcag gaccacttct gcgctcggcc
     8701 cttccggctg gctggtttat tgctgataaa tctggagccg gtgagcgtgg aagccgcggt
     8761 atcattgcag cactggggcc agatggtaag ccctcccgta tcgtagttat ctacacgacg
     8821 gggagtcagg caactatgga tgaacgaaat agacagatcg ctgagatagg tgcctcactg
     8881 attaagcatt ggtaactgtc agaccaagtt tactcatata tactttagat tgatttaaaa
     8941 cttcattttt aatttaaaag gatctaggtg aagatccttt ttgataatct catgaccaaa
     9001 atcccttaac gtgagttttc gttccactga gcgtcagacc ccgtagaaaa gatcaaagga
     9061 tcttcttgag atcctttttt tctgcgcgta atctgctgct tgcaaacaaa aaaaccaccg
     9121 ctaccagcgg tggtttgttt gccggatcaa gagctaccaa ctctttttcc gaaggtaact
     9181 ggcttcagca gagcgcagat accaaatact gttcttctag tgtagccgta gttaggccac
     9241 cacttcaaga actctgtagc accgcctaca tacctcgctc tgctaatcct gttaccagtg
     9301 gctgctgcca gtggcgataa gtcgtgtctt accgggttgg actcaagacg atagttaccg
     9361 gataaggcgc agcggtcggg ctgaacgggg ggttcgtgca cacagcccag cttggagcga
     9421 acgacctaca ccgaactgag atacctacag cgtgagctat gagaaagcgc cacgcttccc
     9481 gaagggagaa aggcggacag gtatccggta agcggcaggg tcggaacagg agagcgcacg
     9541 agggagcttc cagggggaaa cgcctggtat ctttatagtc ctgtcgggtt tcgccacctc
     9601 tgacttgagc gtcgattttt gtgatgctcg tcaggggggc ggagcctatg gaaaaacgcc
     9661 agcaacgcgg cctttttacg gttcctggcc t
//
