LOCUS       sequence-364645-        9821 bp    DNA     circular SYN 15-APR-2025
DEFINITION  expression of gRNA and Cas9 from S. pyogenes with 2A-EGFP.
ACCESSION   .
VERSION     .
KEYWORDS    .
SOURCE      synthetic DNA construct
  ORGANISM  synthetic DNA construct
REFERENCE   1  (bases 1 to 9821)
  AUTHORS   Asimi V, Sampath Kumar A, Niskanen H, Riemenschneider C, Hetzel S, 
            Naderi J, Fasching N, Popitsch N, Du M, Kretzmer H, Smith ZD, 
            Weigert R, Walther M, Mamde S, Meierhofer D, Wittler L, Buschow R, 
            Timmermann B, Cisse II, Ameres SL, Meissner A, Hnisz D
  TITLE     Hijacking of transcriptional condensates by endogenous retroviruses.
  JOURNAL   Nat Genet. 2022 Aug;54(8):1238-1247. doi: 
            10.1038/s41588-022-01132-w. Epub 2022 Jul 21.
  PUBMED    35864192
REFERENCE   2  (bases 1 to 9821)
  AUTHORS   .
  TITLE     Direct Submission
  JOURNAL   Exported Apr 15, 2025 from SnapGene Server 8.0.1
            https://www.snapgene.com
COMMENT     SGRef: number: 1; type: "Journal Article"; doi: 
            "10.1038/s41588-022-01132-w"; journalName: "Nat Genet"; date: 
            "2022-08"; volume: "54"; issue: "8"; pages: "1238-1247"
COMMENT     Sequence Label: pX458-IAP-del-gRNA1
FEATURES             Location/Qualifiers
     source          1..9821
                     /mol_type="other DNA"
                     /organism="synthetic DNA construct"
     rep_origin      162..750
                     /direction=RIGHT
                     /label=ori
                     /note="high-copy-number ColE1/pMB1/pBR322/pUC origin of 
                     replication"
     primer_bind     651..670
                     /label=pBR322ori-F
                     /note="pBR322 origin, forward primer"
     promoter        812..1060
                     /label=U6 promoter
                     /note="RNA polymerase III promoter for human U6 snRNA 
                     (Domitrovich & Kunkel, 2003)"
     primer_bind     812..832
                     /label=hU6-F
                     /note="Human U6 promoter, forward primer"
     primer_bind     983..1002
                     /label=LKO.1 5'
                     /note="Human U6 promoter, forward primer"
     misc_RNA        1082..1157
                     /label=gRNA scaffold
                     /note="guide RNA scaffold for the Streptococcus pyogenes 
                     CRISPR/Cas9 system"
     enhancer        1254..1539
                     /label=CMV enhancer
                     /note="human cytomegalovirus immediate early enhancer; 
                     contains an 18-bp deletion relative to the standard CMV 
                     enhancer"
     intron          1829..2056
                     /label=hybrid intron
                     /note="hybrid between chicken beta-actin (CBA) and minute 
                     virus of mice (MMV) introns (Gray et al., 2011)"
     regulatory      2068..2077
                     /label=Kozak sequence
                     /note="vertebrate consensus sequence for strong initiation 
                     of translation (Kozak, 1987)"
                     /regulatory_class="other"
     CDS             2077..2142
                     /codon_start=1
                     /product="three tandem FLAG(R) epitope tags, followed by an
                     enterokinase cleavage site"
                     /label=3xFLAG
                     /translation="DYKDHDGDYKDHDIDYKDDDDK"
     CDS             2149..2169
                     /codon_start=1
                     /product="nuclear localization signal of SV40 (simian virus
                     40) large T antigen"
                     /label=SV40 NLS
                     /translation="PKKKRKV"
     CDS             2194..6294
                     /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             2194..6294
                     /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             6295..6342
                     /codon_start=1
                     /product="bipartite nuclear localization signal from 
                     nucleoplasmin"
                     /label=nucleoplasmin NLS
                     /translation="KRPAATKKAGQAKKKK"
     CDS             6358..6411
                     /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(6412..6414,6415..7125)
                     /codon_start=1
                     /product="enhanced GFP"
                     /label=EGFP
                     /translation="VSKGEELFTGVVPILVELDGDVNGHKFSVSGEGEGDATYGKLTLK
                     FICTTGKLPVPWPTLVTTLTYGVQCFSRYPDHMKQHDFFKSAMPEGYVQERTIFFKDDG
                     NYKTRAEVKFEGDTLVNRIELKGIDFKEDGNILGHKLEYNYNSHNVYIMADKQKNGIKV
                     NFKIRHNIEDGSVQLADHYQQNTPIGDGPVLLPDNHYLSTQSALSKDPNEKRDHMVLLE
                     FVTAAGITLGMDELYK"
     CDS             join(6412..6414,6415..7125)
                     /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(6715..6734)
                     /label=EXFP-R
                     /note="For distinguishing EGFP variants, reverse primer"
     primer_bind     7062..7083
                     /label=EGFP-C
                     /note="EGFP, forward primer"
     primer_bind     complement(7153..7170)
                     /label=BGH-rev
                     /note="Bovine growth hormone terminator, reverse primer. 
                     Also called BGH reverse"
     polyA_signal    7159..7366
                     /label=bGH poly(A) signal
                     /note="bovine growth hormone polyadenylation signal"
     repeat_region   7375..7515
                     /label=AAV2 ITR
                     /note="inverted terminal repeat of adeno-associated virus 
                     serotype 2"
     repeat_region   7375..7504
                     /label=AAV2 ITR
     gap             7842..7862
                     /estimated_length=21
     primer_bind     complement(7881..7898)
                     /label=M13 Forward
                     /note="In lacZ gene. Also called M13-F20 or M13 (-21) 
                     Forward"
     primer_bind     complement(7881..7897)
                     /label=M13 fwd
                     /note="common sequencing primer, one of multiple similar 
                     variants"
     primer_bind     complement(7890..7912)
                     /label=M13/pUC Forward
                     /note="In lacZ gene"
     rep_origin      8110..8565
                     /direction=RIGHT
                     /label=f1 ori
                     /note="f1 bacteriophage origin of replication; arrow 
                     indicates direction of (+) strand synthesis"
     primer_bind     8407..8428
                     /label=F1ori-F
                     /note="F1 origin, forward primer"
     primer_bind     complement(8582..8601)
                     /label=pRS-marker
                     /note="pRS vectors, use to sequence yeast selectable 
                     marker"
     primer_bind     8701..8723
                     /label=pGEX 3'
                     /note="pGEX vectors, reverse primer"
     primer_bind     complement(8761..8779)
                     /label=pBRforEco
                     /note="pBR322 vectors, upsteam of EcoRI site, forward 
                     primer"
     promoter        8847..8951
                     /gene="bla"
                     /label=AmpR promoter
     CDS             join(8952..9020,9021..9812)
                     /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(9170..9189)
                     /label=Amp-R
                     /note="Ampicillin resistance gene, reverse primer"
ORIGIN
        1 caagtttact catatatact ttagattgat ttaaaacttc atttttaatt taaaaggatc
       61 taggtgaaga tcctttttga taatctcatg accaaaatcc cttaacgtga gttttcgttc
      121 cactgagcgt cagaccccgt agaaaagatc aaaggatctt cttgagatcc tttttttctg
      181 cgcgtaatct gctgcttgca aacaaaaaaa ccaccgctac cagcggtggt ttgtttgccg
      241 gatcaagagc taccaactct ttttccgaag gtaactggct tcagcagagc gcagatacca
      301 aatactgttc ttctagtgta gccgtagtta ggccaccact tcaagaactc tgtagcaccg
      361 cctacatacc tcgctctgct aatcctgtta ccagtggctg ctgccagtgg cgataagtcg
      421 tgtcttaccg ggttggactc aagacgatag ttaccggata aggcgcagcg gtcgggctga
      481 acggggggtt cgtgcacaca gcccagcttg gagcgaacga cctacaccga actgagatac
      541 ctacagcgtg agctatgaga aagcgccacg cttcccgaag ggagaaaggc ggacaggtat
      601 ccggtaagcg gcagggtcgg aacaggagag cgcacgaggg agcttccagg gggaaacgcc
      661 tggtatcttt atagtcctgt cgggtttcgc cacctctgac ttgagcgtcg atttttgtga
      721 tgctcgtcag gggggcggag cctatggaaa aacgccagca acgcggcctt tttacggttc
      781 ctggcctttt gctggccttt tgctcacatg tgagggccta tttcccatga ttccttcata
      841 tttgcatata cgatacaagg ctgttagaga gataattgga attaatttga ctgtaaacac
      901 aaagatatta gtacaaaata cgtgacgtag aaagtaataa tttcttgggt agtttgcagt
      961 tttaaaatta tgttttaaaa tggactatca tatgcttacc gtaacttgaa agtatttcga
     1021 tttcttggct ttatatatct tgtggaaagg acgaaacacc gtgctgtcta atagagaccg
     1081 ggttttagag ctagaaatag caagttaaaa taaggctagt ccgttatcaa cttgaaaaag
     1141 tggcaccgag tcggtgcttt tttgttttag agctagaaat agcaagttaa aataaggcta
     1201 gtccgttttt agcgcgtgcg ccaattctgc agacaaatgg ctctagaggt acccgttaca
     1261 taacttacgg taaatggccc gcctggctga ccgcccaacg acccccgccc attgacgtca
     1321 atagtaacgc caatagggac tttccattga cgtcaatggg tggagtattt acggtaaact
     1381 gcccacttgg cagtacatca agtgtatcat atgccaagta cgccccctat tgacgtcaat
     1441 gacggtaaat ggcccgcctg gcattgtgcc cagtacatga ccttatggga ctttcctact
     1501 tggcagtaca tctacgtatt agtcatcgct attaccatgg tcgaggtgag ccccacgttc
     1561 tgcttcactc tccccatctc ccccccctcc ccacccccaa ttttgtattt atttattttt
     1621 taattatttt gtgcagcgat gggggcgggg gggggggggg gggggsgssg gcgggygsgg
     1681 ggsnggggcg gggcgrgrcr gggggcgggg cggggcgagg cggagaggtg cggcggcagc
     1741 caatcagagc ggcgcgctcc gaaagtttcc ttttatggcg aggcggcggc ggcggcggcc
     1801 ctataaaaag cgaagcgcgc ggcgggcggg agtcgctgcg cgctgccttc gccccgtgcc
     1861 ccgctccgcc gccgcctcgc gccgcccgcc ccggctctga ctgaccgcgt tactcccaca
     1921 ggtgagcggg cgggacggcc cttctcctcc gggctgtaat tagctgagca agaggtaagg
     1981 gtttaaggga tggttggttg gtggggtatt aatgtttaat tacctggagc acctgcctga
     2041 aatcactttt tttcaggttg gaccggtgcc accatggact ataaggacca cgacggagac
     2101 tacaaggatc atgatattga ttacaaagac gatgacgata agatggcccc aaagaagaag
     2161 cggaaggtcg gtatccacgg agtcccagca gccgacaaga agtacagcat cggcctggac
     2221 atcggcacca actctgtggg ctgggccgtg atcaccgacg agtacaaggt gcccagcaag
     2281 aaattcaagg tgctgggcaa caccgaccgg cacagcatca agaagaacct gatcggagcc
     2341 ctgctgttcg acagcggcga aacagccgag gccacccggc tgaagagaac cgccagaaga
     2401 agatacacca gacggaagaa ccggatctgc tatctgcaag agatcttcag caacgagatg
     2461 gccaaggtgg acgacagctt cttccacaga ctggaagagt ccttcctggt ggaagaggat
     2521 aagaagcacg agcggcaccc catcttcggc aacatcgtgg acgaggtggc ctaccacgag
     2581 aagtacccca ccatctacca cctgagaaag aaactggtgg acagcaccga caaggccgac
     2641 ctgcggctga tctatctggc cctggcccac atgatcaagt tccggggcca cttcctgatc
     2701 gagggcgacc tgaaccccga caacagcgac gtggacaagc tgttcatcca gctggtgcag
     2761 acctacaacc agctgttcga ggaaaacccc atcaacgcca gcggcgtgga cgccaaggcc
     2821 atcctgtctg ccagactgag caagagcaga cggctggaaa atctgatcgc ccagctgccc
     2881 ggcgagaaga agaatggcct gttcggaaac ctgattgccc tgagcctggg cctgaccccc
     2941 aacttcaaga gcaacttcga cctggccgag gatgccaaac tgcagctgag caaggacacc
     3001 tacgacgacg acctggacaa cctgctggcc cagatcggcg accagtacgc cgacctgttt
     3061 ctggccgcca agaacctgtc cgacgccatc ctgctgagcg acatcctgag agtgaacacc
     3121 gagatcacca aggcccccct gagcgcctct atgatcaaga gatacgacga gcaccaccag
     3181 gacctgaccc tgctgaaagc tctcgtgcgg cagcagctgc ctgagaagta caaagagatt
     3241 ttcttcgacc agagcaagaa cggctacgcc ggctacattg acggcggagc cagccaggaa
     3301 gagttctaca agttcatcaa gcccatcctg gaaaagatgg acggcaccga ggaactgctc
     3361 gtgaagctga acagagagga cctgctgcgg aagcagcgga ccttcgacaa cggcagcatc
     3421 ccccaccaga tccacctggg agagctgcac gccattctgc ggcggcagga agatttttac
     3481 ccattcctga aggacaaccg ggaaaagatc gagaagatcc tgaccttccg catcccctac
     3541 tacgtgggcc ctctggccag gggaaacagc agattcgcct ggatgaccag aaagagcgag
     3601 gaaaccatca ccccctggaa cttcgaggaa gtggtggaca agggcgcttc cgcccagagc
     3661 ttcatcgagc ggatgaccaa cttcgataag aacctgccca acgagaaggt gctgcccaag
     3721 cacagcctgc tgtacgagta cttcaccgtg tataacgagc tgaccaaagt gaaatacgtg
     3781 accgagggaa tgagaaagcc cgccttcctg agcggcgagc agaaaaaggc catcgtggac
     3841 ctgctgttca agaccaaccg gaaagtgacc gtgaagcagc tgaaagagga ctacttcaag
     3901 aaaatcgagt gcttcgactc cgtggaaatc tccggcgtgg aagatcggtt caacgcctcc
     3961 ctgggcacat accacgatct gctgaaaatt atcaaggaca aggacttcct ggacaatgag
     4021 gaaaacgagg acattctgga agatatcgtg ctgaccctga cactgtttga ggacagagag
     4081 atgatcgagg aacggctgaa aacctatgcc cacctgttcg acgacaaagt gatgaagcag
     4141 ctgaagcggc ggagatacac cggctggggc aggctgagcc ggaagctgat caacggcatc
     4201 cgggacaagc agtccggcaa gacaatcctg gatttcctga agtccgacgg cttcgccaac
     4261 agaaacttca tgcagctgat ccacgacgac agcctgacct ttaaagagga catccagaaa
     4321 gcccaggtgt ccggccaggg cgatagcctg cacgagcaca ttgccaatct ggccggcagc
     4381 cccgccatta agaagggcat cctgcagaca gtgaaggtgg tggacgagct cgtgaaagtg
     4441 atgggccggc acaagcccga gaacatcgtg atcgaaatgg ccagagagaa ccagaccacc
     4501 cagaagggac agaagaacag ccgcgagaga atgaagcgga tcgaagaggg catcaaagag
     4561 ctgggcagcc agatcctgaa agaacacccc gtggaaaaca cccagctgca gaacgagaag
     4621 ctgtacctgt actacctgca gaatgggcgg gatatgtacg tggaccagga actggacatc
     4681 aaccggctgt ccgactacga tgtggaccat atcgtgcctc agagctttct gaaggacgac
     4741 tccatcgaca acaaggtgct gaccagaagc gacaagaacc ggggcaagag cgacaacgtg
     4801 ccctccgaag aggtcgtgaa gaagatgaag aactactggc ggcagctgct gaacgccaag
     4861 ctgattaccc agagaaagtt cgacaatctg accaaggccg agagaggcgg cctgagcgaa
     4921 ctggataagg ccggcttcat caagagacag ctggtggaaa cccggcagat cacaaagcac
     4981 gtggcacaga tcctggactc ccggatgaac actaagtacg acgagaatga caagctgatc
     5041 cgggaagtga aagtgatcac cctgaagtcc aagctggtgt ccgatttccg gaaggatttc
     5101 cagttttaca aagtgcgcga gatcaacaac taccaccacg cccacgacgc ctacctgaac
     5161 gccgtcgtgg gaaccgccct gatcaaaaag taccctaagc tggaaagcga gttcgtgtac
     5221 ggcgactaca aggtgtacga cgtgcggaag atgatcgcca agagcgagca ggaaatcggc
     5281 aaggctaccg ccaagtactt cttctacagc aacatcatga actttttcaa gaccgagatt
     5341 accctggcca acggcgagat ccggaagcgg cctctgatcg agacaaacgg cgaaaccggg
     5401 gagatcgtgt gggataaggg ccgggatttt gccaccgtgc ggaaagtgct gagcatgccc
     5461 caagtgaata tcgtgaaaaa gaccgaggtg cagacaggcg gcttcagcaa agagtctatc
     5521 ctgcccaaga ggaacagcga taagctgatc gccagaaaga aggactggga ccctaagaag
     5581 tacggcggct tcgacagccc caccgtggcc tattctgtgc tggtggtggc caaagtggaa
     5641 aagggcaagt ccaagaaact gaagagtgtg aaagagctgc tggggatcac catcatggaa
     5701 agaagcagct tcgagaagaa tcccatcgac tttctggaag ccaagggcta caaagaagtg
     5761 aaaaaggacc tgatcatcaa gctgcctaag tactccctgt tcgagctgga aaacggccgg
     5821 aagagaatgc tggcctctgc cggcgaactg cagaagggaa acgaactggc cctgccctcc
     5881 aaatatgtga acttcctgta cctggccagc cactatgaga agctgaaggg ctcccccgag
     5941 gataatgagc agaaacagct gtttgtggaa cagcacaagc actacctgga cgagatcatc
     6001 gagcagatca gcgagttctc caagagagtg atcctggccg acgctaatct ggacaaagtg
     6061 ctgtccgcct acaacaagca ccgggataag cccatcagag agcaggccga gaatatcatc
     6121 cacctgttta ccctgaccaa tctgggagcc cctgccgcct tcaagtactt tgacaccacc
     6181 atcgaccgga agaggtacac cagcaccaaa gaggtgctgg acgccaccct gatccaccag
     6241 agcatcaccg gcctgtacga gacacggatc gacctgtctc agctgggagg cgacaaaagg
     6301 ccggcggcca cgaaaaaggc cggccaggca aaaaagaaaa aggaattcgg cagtggagag
     6361 ggcagaggaa gtctgctaac atgcggtgac gtcgaggaga atcctggccc agtgagcaag
     6421 ggcgaggagc tgttcaccgg ggtggtgccc atcctggtcg agctggacgg cgacgtaaac
     6481 ggccacaagt tcagcgtgtc cggcgagggc gagggcgatg ccacctacgg caagctgacc
     6541 ctgaagttca tctgcaccac cggcaagctg cccgtgccct ggcccaccct cgtgaccacc
     6601 ctgacctacg gcgtgcagtg cttcagccgc taccccgacc acatgaagca gcacgacttc
     6661 ttcaagtccg ccatgcccga aggctacgtc caggagcgca ccatcttctt caaggacgac
     6721 ggcaactaca agacccgcgc cgaggtgaag ttcgagggcg acaccctggt gaaccgcatc
     6781 gagctgaagg gcatcgactt caaggaggac ggcaacatcc tggggcacaa gctggagtac
     6841 aactacaaca gccacaacgt ctatatcatg gccgacaagc agaagaacgg catcaaggtg
     6901 aacttcaaga tccgccacaa catcgaggac ggcagcgtgc agctcgccga ccactaccag
     6961 cagaacaccc ccatcggcga cggccccgtg ctgctgcccg acaaccacta cctgagcacc
     7021 cagtccgccc tgagcaaaga ccccaacgag aagcgcgatc acatggtcct gctggagttc
     7081 gtgaccgccg ccgggatcac tctcggcatg gacgagctgt acaaggaatt ctaactagag
     7141 ctcgctgatc agcctcgact gtgccttcta gttgccagcc atctgttgtt tgcccctccc
     7201 ccgtgccttc cttgaccctg gaaggtgcca ctcccactgt cctttcctaa taaaatgagg
     7261 aaattgcatc gcattgtctg agtaggtgtc attctattct ggggggtggg gtggggcagg
     7321 acagcaaggg ggaggattgg gaagagaata gcaggcatgc tggggagcgg ccgcaggaac
     7381 ccctagtgat ggagttggcc actccctctc tgcgcgctcg ctcgctcact gaggccgggc
     7441 gaccaaaggt cgcccgacgc ccgggctttg cccgggcggc ctcagtgagc gagcgagcgc
     7501 gcagctgcct gcaggggcgc ctgatgcggt attttctcct tacgcatctg tgcggtattt
     7561 cacaccgcat acgtcaaagc aaccatagta cgcgccctgt agcggcgcat taagcgcggc
     7621 gggtgtggtg gttacgcgca gcgtgaccgc tacacttgcc agcgccttag cgcccgctcc
     7681 tttcgctttc ttcccttcct ttctcgccac gttcgccggc tttccccgtc aagctctaaa
     7741 tcgggggctc cctttagggt tccgatttag tgctttacgg cacctcgacc ccaaaaaact
     7801 tgatttgggt gatggttcac gtagtgggcc atcgccctga tnnnnnnnnn nnnnnnnnnn
     7861 nngtcgtatt acgcgcgctc actggccgtc gttttacaac gtcgtgactg ggaaaaccct
     7921 ggcgttaccc aacttaatcg ccttgcagca catccccctt tcgccagctg gcgtaatagc
     7981 gaagaggccc gcaccgatcg cccttcccaa cagttgcgca gcctgaaykg cnarkggcgc
     8041 ctgatgcggt attttctcct tacgcatctg tgcggtattt cacaccgcat acgtcaaagc
     8101 aaccatagta cgcgccctgt agcggcgcat taagcgcggc gggtgtggtg gttacgcgca
     8161 gcgtgaccgc tacacttgcc agcgccttag cgcccgctcc tttcgctttc ttcccttcct
     8221 ttctcgccac gttcgccggc tttccccgtc aagctctaaa tcgggggctc cctttagggt
     8281 tccgatttag tgctttacgg cacctcgacc ccaaaaaact tgatttgggt gatggttcac
     8341 gtagtgggcc atcgccctga tagacggttt ttcgcccttt gacgttggag tccacgttct
     8401 ttaatagtgg actcttgttc caaactggaa caacactcaa ctctatctcg ggctattctt
     8461 ttgatttata agggattttg ccgatttcgg tctattggtt aaaaaatgag ctgatttaac
     8521 aaaaatttaa cgcgaatttt aacaaaatat taacgtttac aattttatgg tgcactctca
     8581 gtacaatctg ctctgatgcc gcatagttaa gccagccccg acacccgcca acacccgctg
     8641 acgcgccctg acgggcttgt ctgctcccgg catccgctta cagacaagct gtgaccgtct
     8701 ccgggagctg catgtgtcag aggttttcac cgtcatcacc gaaacgcgcg agacgaaagg
     8761 gcctcgtgat acgcctattt ttataggtta atgtcatgat aataatggtt tcttagacgt
     8821 caggtggcac ttttcgggga aatgtgcgcg gaacccctat ttgtttattt ttctaaatac
     8881 attcaaatat gtatccgctc atgagacaat aaccctgata aatgcttcaa taatattgaa
     8941 aaaggaagag tatgagtatt caacatttcc gtgtcgccct tattcccttt tttgcggcat
     9001 tttgccttcc tgtttttgct cacccagaaa cgctggtgaa agtaaaagat gctgaagatc
     9061 agttgggtgc acgagtgggt tacatcgaac tggatctcaa cagcggtaag atccttgaga
     9121 gttttcgccc cgaagaacgt tttccaatga tgagcacttt taaagttctg ctatgtggcg
     9181 cggtattatc ccgtattgac gccgggcaag agcaactcgg tcgccgcata cactattctc
     9241 agaatgactt ggttgagtac tcaccagtca cagaaaagca tcttacggat ggcatgacag
     9301 taagagaatt atgcagtgct gccataacca tgagtgataa cactgcggcc aacttacttc
     9361 tgacaacgat cggaggaccg aaggagctaa ccgctttttt gcacaacatg ggggatcatg
     9421 taactcgcct tgatcgttgg gaaccggagc tgaatgaagc cataccaaac gacgagcgtg
     9481 acaccacgat gcctgtagca atggcaacaa cgttgcgcaa actattaact ggcgaactac
     9541 ttactctagc ttcccggcaa caattaatag actggatgga ggcggataaa gttgcaggac
     9601 cacttctgcg ctcggccctt ccggctggct ggtttattgc tgataaatct ggagccggtg
     9661 agcgtggaag ccgcggtatc attgcagcac tggggccaga tggtaagccc tcccgtatcg
     9721 tagttatcta cacgacgggg agtcaggcaa ctatggatga acgaaataga cagatcgctg
     9781 agataggtgc ctcactgatt aagcattggt aactgtcaga c
//
