LOCUS       sequence-329092-        9275 bp    DNA     circular SYN 14-APR-2025
DEFINITION  Transcription of TREX1 guide RNA and Cas9 expression for CRISPR/Cas9
            in mammalian cells.
ACCESSION   .
VERSION     .
KEYWORDS    .
SOURCE      synthetic DNA construct
  ORGANISM  synthetic DNA construct
REFERENCE   1  (bases 1 to 9275)
  AUTHORS   Mohr L, Toufektchan E, von Morgen P, Chu K, Kapoor A, Maciejowski J
  TITLE     ER-directed TREX1 limits cGAS activation at micronuclei.
  JOURNAL   Mol Cell. 2021 Jan 12. pii: S1097-2765(20)30958-8. doi: 
            10.1016/j.molcel.2020.12.037.
  PUBMED    33476576
REFERENCE   2  (bases 1 to 9275)
  AUTHORS   .
  TITLE     Direct Submission
  JOURNAL   Exported Apr 14, 2025 from SnapGene Server 8.0.1
            https://www.snapgene.com
COMMENT     SGRef: number: 1; type: "Journal Article"; journalName: "Mol Cell. 
            2021 Jan 12. pii: S1097-2765(20)30958-8. doi: 
            10.1016/j.molcel.2020.12.037."
COMMENT     Sequence Label: pU6-TREX1 g1 Cas9-T2A-mCherry
FEATURES             Location/Qualifiers
     source          1..9275
                     /mol_type="other DNA"
                     /organism="synthetic DNA construct"
     promoter        1..249
                     /label=U6 promoter
                     /note="RNA polymerase III promoter for human U6 snRNA 
                     (Domitrovich & Kunkel, 2003)"
     primer_bind     1..21
                     /label=hU6-F
                     /note="Human U6 promoter, forward primer"
     primer_bind     172..191
                     /label=LKO.1 5'
                     /note="Human U6 promoter, forward primer"
     misc_RNA        267..342
                     /label=gRNA scaffold
                     /note="guide RNA scaffold for the Streptococcus pyogenes 
                     CRISPR/Cas9 system"
     enhancer        439..724
                     /label=CMV enhancer
                     /note="human cytomegalovirus immediate early enhancer; 
                     contains an 18-bp deletion relative to the standard CMV 
                     enhancer"
     promoter        726..1003
                     /label=chicken beta-actin promoter
     intron          1004..1231
                     /label=hybrid intron
                     /note="hybrid between chicken beta-actin (CBA) and minute 
                     virus of mice (MMV) introns (Gray et al., 2011)"
     regulatory      1243..1252
                     /label=Kozak sequence
                     /note="vertebrate consensus sequence for strong initiation 
                     of translation (Kozak, 1987)"
                     /regulatory_class="other"
     CDS             1252..1317
                     /codon_start=1
                     /product="three tandem FLAG(R) epitope tags, followed by an
                     enterokinase cleavage site"
                     /label=3xFLAG
                     /translation="DYKDHDGDYKDHDIDYKDDDDK"
     CDS             1324..1344
                     /codon_start=1
                     /product="nuclear localization signal of SV40 (simian virus
                     40) large T antigen"
                     /label=SV40 NLS
                     /translation="PKKKRKV"
     CDS             1369..5469
                     /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             1369..5469
                     /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             5470..5517
                     /codon_start=1
                     /product="bipartite nuclear localization signal from 
                     nucleoplasmin"
                     /label=nucleoplasmin NLS
                     /translation="KRPAATKKAGQAKKKK"
     CDS             5521..5574
                     /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             5581..6291
                     /codon_start=1
                     /product="monomeric derivative of DsRed fluorescent protein
                     (Shaner et al., 2004)"
                     /label=mCherry
                     /note="mammalian codon-optimized"
                     /translation="MVSKGEEDNMAIIKEFMRFKVHMEGSVNGHEFEIEGEGEGRPYEG
                     TQTAKLKVTKGGPLPFAWDILSPQFMYGSKAYVKHPADIPDYLKLSFPEGFKWERVMNF
                     EDGGVVTVTQDSSLQDGEFIYKVKLRGTNFPSDGPVMQKKTMGWEASSERMYPEDGALK
                     GEIKQRLKLKDGGHYDAEVKTTYKAKKPVQLPGAYNVNIKLDITSHNEDYTIVEQYERA
                     EGRHSTGGMDELYK"
     primer_bind     complement(5726..5744)
                     /label=mCherry-R
                     /note="mCherry, reverse primer"
     primer_bind     complement(6316..6333)
                     /label=BGH-rev
                     /note="Bovine growth hormone terminator, reverse primer. 
                     Also called BGH reverse"
     polyA_signal    6322..6529
                     /label=bGH poly(A) signal
                     /note="bovine growth hormone polyadenylation signal"
     repeat_region   6538..6678
                     /label=AAV2 ITR
                     /note="inverted terminal repeat of adeno-associated virus 
                     serotype 2"
     repeat_region   6538..6667
                     /label=AAV2 ITR
     rep_origin      6753..7208
                     /direction=RIGHT
                     /label=f1 ori
                     /note="f1 bacteriophage origin of replication; arrow 
                     indicates direction of (+) strand synthesis"
     primer_bind     complement(6840..6859)
                     /label=F1ori-R
                     /note="F1 origin, reverse primer"
     primer_bind     7050..7071
                     /label=F1ori-F
                     /note="F1 origin, forward primer"
     primer_bind     complement(7225..7244)
                     /label=pRS-marker
                     /note="pRS vectors, use to sequence yeast selectable 
                     marker"
     primer_bind     7344..7366
                     /label=pGEX 3'
                     /note="pGEX vectors, reverse primer"
     primer_bind     complement(7404..7422)
                     /label=pBRforEco
                     /note="pBR322 vectors, upsteam of EcoRI site, forward 
                     primer"
     promoter        7490..7594
                     /gene="bla"
                     /label=AmpR promoter
     CDS             join(7595..7663,7664..8455)
                     /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(7813..7832)
                     /label=Amp-R
                     /note="Ampicillin resistance gene, reverse primer"
     rep_origin      8626..9214
                     /direction=RIGHT
                     /label=ori
                     /note="high-copy-number ColE1/pMB1/pBR322/pUC origin of 
                     replication"
     primer_bind     9115..9134
                     /label=pBR322ori-F
                     /note="pBR322 origin, forward primer"
ORIGIN
        1 gagggcctat ttcccatgat tccttcatat ttgcatatac gatacaaggc tgttagagag
       61 ataattggaa ttaatttgac tgtaaacaca aagatattag tacaaaatac gtgacgtaga
      121 aagtaataat ttcttgggta gtttgcagtt ttaaaattat gttttaaaat ggactatcat
      181 atgcttaccg taacttgaaa gtatttcgat ttcttggctt tatatatctt gtggaaagga
      241 cgaaacaccg agccccccca cctctcgttt tagagctaga aatagcaagt taaaataagg
      301 ctagtccgtt atcaacttga aaaagtggca ccgagtcggt gcttttttgt tttagagcta
      361 gaaatagcaa gttaaaataa ggctagtccg tttttagcgc gtgcgccaat tctgcagaca
      421 aatggctcta gaggtacccg ttacataact tacggtaaat ggcccgcctg gctgaccgcc
      481 caacgacccc cgcccattga cgtcaatagt aacgccaata gggactttcc attgacgtca
      541 atgggtggag tatttacggt aaactgccca cttggcagta catcaagtgt atcatatgcc
      601 aagtacgccc cctattgacg tcaatgacgg taaatggccc gcctggcatt gtgcccagta
      661 catgacctta tgggactttc ctacttggca gtacatctac gtattagtca tcgctattac
      721 catggtcgag gtgagcccca cgttctgctt cactctcccc atctcccccc cctccccacc
      781 cccaattttg tatttattta ttttttaatt attttgtgca gcgatggggg cggggggggg
      841 gggggggcgc gcgccaggcg gggcggggcg gggcgagggg cggggcgggg cgaggcggag
      901 aggtgcggcg gcagccaatc agagcggcgc gctccgaaag tttcctttta tggcgaggcg
      961 gcggcggcgg cggccctata aaaagcgaag cgcgcggcgg gcgggagtcg ctgcgcgctg
     1021 ccttcgcccc gtgccccgct ccgccgccgc ctcgcgccgc ccgccccggc tctgactgac
     1081 cgcgttactc ccacaggtga gcgggcggga cggcccttct cctccgggct gtaattagct
     1141 gagcaagagg taagggttta agggatggtt ggttggtggg gtattaatgt ttaattacct
     1201 ggagcacctg cctgaaatca ctttttttca ggttggaccg gtgccaccat ggactataag
     1261 gaccacgacg gagactacaa ggatcatgat attgattaca aagacgatga cgataagatg
     1321 gccccaaaga agaagcggaa ggtcggtatc cacggagtcc cagcagccga caagaagtac
     1381 agcatcggcc tggacatcgg caccaactct gtgggctggg ccgtgatcac cgacgagtac
     1441 aaggtgccca gcaagaaatt caaggtgctg ggcaacaccg accggcacag catcaagaag
     1501 aacctgatcg gagccctgct gttcgacagc ggcgaaacag ccgaggccac ccggctgaag
     1561 agaaccgcca gaagaagata caccagacgg aagaaccgga tctgctatct gcaagagatc
     1621 ttcagcaacg agatggccaa ggtggacgac agcttcttcc acagactgga agagtccttc
     1681 ctggtggaag aggataagaa gcacgagcgg caccccatct tcggcaacat cgtggacgag
     1741 gtggcctacc acgagaagta ccccaccatc taccacctga gaaagaaact ggtggacagc
     1801 accgacaagg ccgacctgcg gctgatctat ctggccctgg cccacatgat caagttccgg
     1861 ggccacttcc tgatcgaggg cgacctgaac cccgacaaca gcgacgtgga caagctgttc
     1921 atccagctgg tgcagaccta caaccagctg ttcgaggaaa accccatcaa cgccagcggc
     1981 gtggacgcca aggccatcct gtctgccaga ctgagcaaga gcagacggct ggaaaatctg
     2041 atcgcccagc tgcccggcga gaagaagaat ggcctgttcg gaaacctgat tgccctgagc
     2101 ctgggcctga cccccaactt caagagcaac ttcgacctgg ccgaggatgc caaactgcag
     2161 ctgagcaagg acacctacga cgacgacctg gacaacctgc tggcccagat cggcgaccag
     2221 tacgccgacc tgtttctggc cgccaagaac ctgtccgacg ccatcctgct gagcgacatc
     2281 ctgagagtga acaccgagat caccaaggcc cccctgagcg cctctatgat caagagatac
     2341 gacgagcacc accaggacct gaccctgctg aaagctctcg tgcggcagca gctgcctgag
     2401 aagtacaaag agattttctt cgaccagagc aagaacggct acgccggcta cattgacggc
     2461 ggagccagcc aggaagagtt ctacaagttc atcaagccca tcctggaaaa gatggacggc
     2521 accgaggaac tgctcgtgaa gctgaacaga gaggacctgc tgcggaagca gcggaccttc
     2581 gacaacggca gcatccccca ccagatccac ctgggagagc tgcacgccat tctgcggcgg
     2641 caggaagatt tttacccatt cctgaaggac aaccgggaaa agatcgagaa gatcctgacc
     2701 ttccgcatcc cctactacgt gggccctctg gccaggggaa acagcagatt cgcctggatg
     2761 accagaaaga gcgaggaaac catcaccccc tggaacttcg aggaagtggt ggacaagggc
     2821 gcttccgccc agagcttcat cgagcggatg accaacttcg ataagaacct gcccaacgag
     2881 aaggtgctgc ccaagcacag cctgctgtac gagtacttca ccgtgtataa cgagctgacc
     2941 aaagtgaaat acgtgaccga gggaatgaga aagcccgcct tcctgagcgg cgagcagaaa
     3001 aaggccatcg tggacctgct gttcaagacc aaccggaaag tgaccgtgaa gcagctgaaa
     3061 gaggactact tcaagaaaat cgagtgcttc gactccgtgg aaatctccgg cgtggaagat
     3121 cggttcaacg cctccctggg cacataccac gatctgctga aaattatcaa ggacaaggac
     3181 ttcctggaca atgaggaaaa cgaggacatt ctggaagata tcgtgctgac cctgacactg
     3241 tttgaggaca gagagatgat cgaggaacgg ctgaaaacct atgcccacct gttcgacgac
     3301 aaagtgatga agcagctgaa gcggcggaga tacaccggct ggggcaggct gagccggaag
     3361 ctgatcaacg gcatccggga caagcagtcc ggcaagacaa tcctggattt cctgaagtcc
     3421 gacggcttcg ccaacagaaa cttcatgcag ctgatccacg acgacagcct gacctttaaa
     3481 gaggacatcc agaaagccca ggtgtccggc cagggcgata gcctgcacga gcacattgcc
     3541 aatctggccg gcagccccgc cattaagaag ggcatcctgc agacagtgaa ggtggtggac
     3601 gagctcgtga aagtgatggg ccggcacaag cccgagaaca tcgtgatcga aatggccaga
     3661 gagaaccaga ccacccagaa gggacagaag aacagccgcg agagaatgaa gcggatcgaa
     3721 gagggcatca aagagctggg cagccagatc ctgaaagaac accccgtgga aaacacccag
     3781 ctgcagaacg agaagctgta cctgtactac ctgcagaatg ggcgggatat gtacgtggac
     3841 caggaactgg acatcaaccg gctgtccgac tacgatgtgg accatatcgt gcctcagagc
     3901 tttctgaagg acgactccat cgacaacaag gtgctgacca gaagcgacaa gaaccggggc
     3961 aagagcgaca acgtgccctc cgaagaggtc gtgaagaaga tgaagaacta ctggcggcag
     4021 ctgctgaacg ccaagctgat tacccagaga aagttcgaca atctgaccaa ggccgagaga
     4081 ggcggcctga gcgaactgga taaggccggc ttcatcaaga gacagctggt ggaaacccgg
     4141 cagatcacaa agcacgtggc acagatcctg gactcccgga tgaacactaa gtacgacgag
     4201 aatgacaagc tgatccggga agtgaaagtg atcaccctga agtccaagct ggtgtccgat
     4261 ttccggaagg atttccagtt ttacaaagtg cgcgagatca acaactacca ccacgcccac
     4321 gacgcctacc tgaacgccgt cgtgggaacc gccctgatca aaaagtaccc taagctggaa
     4381 agcgagttcg tgtacggcga ctacaaggtg tacgacgtgc ggaagatgat cgccaagagc
     4441 gagcaggaaa tcggcaaggc taccgccaag tacttcttct acagcaacat catgaacttt
     4501 ttcaagaccg agattaccct ggccaacggc gagatccgga agcggcctct gatcgagaca
     4561 aacggcgaaa ccggggagat cgtgtgggat aagggccggg attttgccac cgtgcggaaa
     4621 gtgctgagca tgccccaagt gaatatcgtg aaaaagaccg aggtgcagac aggcggcttc
     4681 agcaaagagt ctatcctgcc caagaggaac agcgataagc tgatcgccag aaagaaggac
     4741 tgggacccta agaagtacgg cggcttcgac agccccaccg tggcctattc tgtgctggtg
     4801 gtggccaaag tggaaaaggg caagtccaag aaactgaaga gtgtgaaaga gctgctgggg
     4861 atcaccatca tggaaagaag cagcttcgag aagaatccca tcgactttct ggaagccaag
     4921 ggctacaaag aagtgaaaaa ggacctgatc atcaagctgc ctaagtactc cctgttcgag
     4981 ctggaaaacg gccggaagag aatgctggcc tctgccggcg aactgcagaa gggaaacgaa
     5041 ctggccctgc cctccaaata tgtgaacttc ctgtacctgg ccagccacta tgagaagctg
     5101 aagggctccc ccgaggataa tgagcagaaa cagctgtttg tggaacagca caagcactac
     5161 ctggacgaga tcatcgagca gatcagcgag ttctccaaga gagtgatcct ggccgacgct
     5221 aatctggaca aagtgctgtc cgcctacaac aagcaccggg ataagcccat cagagagcag
     5281 gccgagaata tcatccacct gtttaccctg accaatctgg gagcccctgc cgccttcaag
     5341 tactttgaca ccaccatcga ccggaagagg tacaccagca ccaaagaggt gctggacgcc
     5401 accctgatcc accagagcat caccggcctg tacgagacac ggatcgacct gtctcagctg
     5461 ggaggcgaca aaaggccggc ggccacgaaa aaggccggcc aggcaaaaaa gaaaaagctt
     5521 gagggcagag gaagtctgct aacatgcggt gacgtggagg agaatcccgg ccctgctagc
     5581 atggtgagca agggcgagga ggataacatg gccatcatca aggagttcat gcgcttcaag
     5641 gtgcacatgg agggctccgt gaacggccac gagttcgaga tcgagggcga gggcgagggc
     5701 cgcccctacg agggcaccca gaccgccaag ctgaaggtga ccaagggtgg ccccctgccc
     5761 ttcgcctggg acatcctgtc ccctcagttc atgtacggct ccaaggccta cgtgaagcac
     5821 cccgccgaca tccccgacta cttgaagctg tccttccccg agggcttcaa gtgggagcgc
     5881 gtgatgaact tcgaggacgg cggcgtggtg accgtgaccc aggactcctc cctgcaggac
     5941 ggcgagttca tctacaaggt gaagctgcgc ggcaccaact tcccctcaga cggccccgta
     6001 atgcagaaga aaaccatggg ctgggaggcc tcctccgagc ggatgtaccc cgaggacggc
     6061 gccctgaagg gcgagatcaa gcagaggctg aagctgaagg acggcggcca ctacgacgct
     6121 gaggtcaaga ccacctacaa ggccaagaag cccgtgcagc tgcccggcgc ctacaacgtc
     6181 aacatcaagt tggacatcac ctcccacaac gaggactaca ccatcgtgga acagtacgaa
     6241 cgcgccgagg gccgccactc caccggcggc atggacgagc tgtacaagta agaattccta
     6301 gagctcgctg atcagcctcg actgtgcctt ctagttgcca gccatctgtt gtttgcccct
     6361 cccccgtgcc ttccttgacc ctggaaggtg ccactcccac tgtcctttcc taataaaatg
     6421 aggaaattgc atcgcattgt ctgagtaggt gtcattctat tctggggggt ggggtggggc
     6481 aggacagcaa gggggaggat tgggaagaga atagcaggca tgctggggag cggccgcagg
     6541 aacccctagt gatggagttg gccactccct ctctgcgcgc tcgctcgctc actgaggccg
     6601 ggcgaccaaa ggtcgcccga cgcccgggct ttgcccgggc ggcctcagtg agcgagcgag
     6661 cgcgcagctg cctgcagggg cgcctgatgc ggtattttct ccttacgcat ctgtgcggta
     6721 tttcacaccg catacgtcaa agcaaccata gtacgcgccc tgtagcggcg cattaagcgc
     6781 ggcgggtgtg gtggttacgc gcagcgtgac cgctacactt gccagcgcct tagcgcccgc
     6841 tcctttcgct ttcttccctt cctttctcgc cacgttcgcc ggctttcccc gtcaagctct
     6901 aaatcggggg ctccctttag ggttccgatt tagtgcttta cggcacctcg accccaaaaa
     6961 acttgatttg ggtgatggtt cacgtagtgg gccatcgccc tgatagacgg tttttcgccc
     7021 tttgacgttg gagtccacgt tctttaatag tggactcttg ttccaaactg gaacaacact
     7081 caactctatc tcgggctatt cttttgattt ataagggatt ttgccgattt cggtctattg
     7141 gttaaaaaat gagctgattt aacaaaaatt taacgcgaat tttaacaaaa tattaacgtt
     7201 tacaatttta tggtgcactc tcagtacaat ctgctctgat gccgcatagt taagccagcc
     7261 ccgacacccg ccaacacccg ctgacgcgcc ctgacgggct tgtctgctcc cggcatccgc
     7321 ttacagacaa gctgtgaccg tctccgggag ctgcatgtgt cagaggtttt caccgtcatc
     7381 accgaaacgc gcgagacgaa agggcctcgt gatacgccta tttttatagg ttaatgtcat
     7441 gataataatg gtttcttaga cgtcaggtgg cacttttcgg ggaaatgtgc gcggaacccc
     7501 tatttgttta tttttctaaa tacattcaaa tatgtatccg ctcatgagac aataaccctg
     7561 ataaatgctt caataatatt gaaaaaggaa gagtatgagt attcaacatt tccgtgtcgc
     7621 ccttattccc ttttttgcgg cattttgcct tcctgttttt gctcacccag aaacgctggt
     7681 gaaagtaaaa gatgctgaag atcagttggg tgcacgagtg ggttacatcg aactggatct
     7741 caacagcggt aagatccttg agagttttcg ccccgaagaa cgttttccaa tgatgagcac
     7801 ttttaaagtt ctgctatgtg gcgcggtatt atcccgtatt gacgccgggc aagagcaact
     7861 cggtcgccgc atacactatt ctcagaatga cttggttgag tactcaccag tcacagaaaa
     7921 gcatcttacg gatggcatga cagtaagaga attatgcagt gctgccataa ccatgagtga
     7981 taacactgcg gccaacttac ttctgacaac gatcggagga ccgaaggagc taaccgcttt
     8041 tttgcacaac atgggggatc atgtaactcg ccttgatcgt tgggaaccgg agctgaatga
     8101 agccatacca aacgacgagc gtgacaccac gatgcctgta gcaatggcaa caacgttgcg
     8161 caaactatta actggcgaac tacttactct agcttcccgg caacaattaa tagactggat
     8221 ggaggcggat aaagttgcag gaccacttct gcgctcggcc cttccggctg gctggtttat
     8281 tgctgataaa tctggagccg gtgagcgtgg aagccgcggt atcattgcag cactggggcc
     8341 agatggtaag ccctcccgta tcgtagttat ctacacgacg gggagtcagg caactatgga
     8401 tgaacgaaat agacagatcg ctgagatagg tgcctcactg attaagcatt ggtaactgtc
     8461 agaccaagtt tactcatata tactttagat tgatttaaaa cttcattttt aatttaaaag
     8521 gatctaggtg aagatccttt ttgataatct catgaccaaa atcccttaac gtgagttttc
     8581 gttccactga gcgtcagacc ccgtagaaaa gatcaaagga tcttcttgag atcctttttt
     8641 tctgcgcgta atctgctgct tgcaaacaaa aaaaccaccg ctaccagcgg tggtttgttt
     8701 gccggatcaa gagctaccaa ctctttttcc gaaggtaact ggcttcagca gagcgcagat
     8761 accaaatact gttcttctag tgtagccgta gttaggccac cacttcaaga actctgtagc
     8821 accgcctaca tacctcgctc tgctaatcct gttaccagtg gctgctgcca gtggcgataa
     8881 gtcgtgtctt accgggttgg actcaagacg atagttaccg gataaggcgc agcggtcggg
     8941 ctgaacgggg ggttcgtgca cacagcccag cttggagcga acgacctaca ccgaactgag
     9001 atacctacag cgtgagctat gagaaagcgc cacgcttccc gaagggagaa aggcggacag
     9061 gtatccggta agcggcaggg tcggaacagg agagcgcacg agggagcttc cagggggaaa
     9121 cgcctggtat ctttatagtc ctgtcgggtt tcgccacctc tgacttgagc gtcgattttt
     9181 gtgatgctcg tcaggggggc ggagcctatg gaaaaacgcc agcaacgcgg cctttttacg
     9241 gttcctggcc ttttgctggc cttttgctca catgt
//
