LOCUS       sequence-324929-        9722 bp    DNA     circular SYN 15-DEC-2025
DEFINITION  Plasmid encoding for 2 gRNAs targeting the human BAX gene and a CMV 
            driven nuclease Cas9 followed by self-cleaving EGFP.
ACCESSION   .
VERSION     .
KEYWORDS    .
SOURCE      synthetic DNA construct
  ORGANISM  synthetic DNA construct
REFERENCE   1  (bases 1 to 9722)
  AUTHORS   Tigano M, Vargas DC, Tremblay-Belzile S, Fu Y, Sfeir A
  TITLE     Nuclear sensing of breaks in mitochondrial DNA enhances immune 
            surveillance.
  JOURNAL   Nature. 2021 Mar;591(7850):477-481. doi: 10.1038/s41586-021-03269-w.
            Epub 2021 Feb 24.
  PUBMED    33627873
REFERENCE   2  (bases 1 to 9722)
  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"; doi: 
            "10.1038/s41586-021-03269-w"; journalName: "Nature"; date: 
            "2021-03"; volume: "591"; issue: "7850"; pages: "477-481"
COMMENT     Sequence Label: pSpCas9nucl-T2A-EGFP - BAXgRNA1- BAXgRNA2
FEATURES             Location/Qualifiers
     source          1..9722
                     /mol_type="other DNA"
                     /organism="synthetic DNA construct"
     rep_origin      8..596
                     /direction=RIGHT
                     /label=ori
                     /note="high-copy-number ColE1/pMB1/pBR322/pUC origin of 
                     replication"
     primer_bind     497..516
                     /label=pBR322ori-F
                     /note="pBR322 origin, forward primer"
     promoter        658..906
                     /label=U6 promoter
                     /note="RNA polymerase III promoter for human U6 snRNA 
                     (Domitrovich & Kunkel, 2003)"
     misc_RNA        928..1003
                     /label=gRNA scaffold
                     /note="guide RNA scaffold for the Streptococcus pyogenes 
                     CRISPR/Cas9 system"
     promoter        1094..1342
                     /label=U6 promoter
                     /note="RNA polymerase III promoter for human U6 snRNA 
                     (Domitrovich & Kunkel, 2003)"
     misc_RNA        1364..1439
                     /label=gRNA scaffold
                     /note="guide RNA scaffold for the Streptococcus pyogenes 
                     CRISPR/Cas9 system"
     enhancer        1530..1815
                     /label=CMV enhancer
                     /note="human cytomegalovirus immediate early enhancer; 
                     contains an 18-bp deletion relative to the standard CMV 
                     enhancer"
     promoter        1817..2094
                     /label=chicken beta-actin promoter
     intron          2095..2323
                     /label=hybrid intron
                     /note="hybrid between chicken beta-actin (CBA) and minute 
                     virus of mice (MMV) introns (Gray et al., 2011)"
     regulatory      2335..2344
                     /label=Kozak sequence
                     /note="vertebrate consensus sequence for strong initiation 
                     of translation (Kozak, 1987)"
                     /regulatory_class="other"
     CDS             2344..2409
                     /codon_start=1
                     /product="three tandem FLAG(R) epitope tags, followed by an
                     enterokinase cleavage site"
                     /label=3xFLAG
                     /translation="DYKDHDGDYKDHDIDYKDDDDK"
     CDS             2416..2436
                     /codon_start=1
                     /product="nuclear localization signal of SV40 (simian virus
                     40) large T antigen"
                     /label=SV40 NLS
                     /translation="PKKKRKV"
     CDS             2461..6561
                     /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             2461..6561
                     /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             6562..6609
                     /codon_start=1
                     /product="bipartite nuclear localization signal from 
                     nucleoplasmin"
                     /label=nucleoplasmin NLS
                     /translation="KRPAATKKAGQAKKKK"
     CDS             6625..6678
                     /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(6679..6681,6682..7392)
                     /codon_start=1
                     /product="enhanced GFP"
                     /label=EGFP
                     /translation="VSKGEELFTGVVPILVELDGDVNGHKFSVSGEGEGDATYGKLTLK
                     FICTTGKLPVPWPTLVTTLTYGVQCFSRYPDHMKQHDFFKSAMPEGYVQERTIFFKDDG
                     NYKTRAEVKFEGDTLVNRIELKGIDFKEDGNILGHKLEYNYNSHNVYIMADKQKNGIKV
                     NFKIRHNIEDGSVQLADHYQQNTPIGDGPVLLPDNHYLSTQSALSKDPNEKRDHMVLLE
                     FVTAAGITLGMDELYK"
     CDS             join(6679..6681,6682..7392)
                     /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(6982..7001)
                     /label=EXFP-R
                     /note="For distinguishing EGFP variants, reverse primer"
     primer_bind     7329..7350
                     /label=EGFP-C
                     /note="EGFP, forward primer"
     primer_bind     complement(7420..7437)
                     /label=BGH-rev
                     /note="Bovine growth hormone terminator, reverse primer. 
                     Also called BGH reverse"
     polyA_signal    7426..7633
                     /label=bGH poly(A) signal
                     /note="bovine growth hormone polyadenylation signal"
     repeat_region   7642..7782
                     /label=AAV2 ITR
                     /note="inverted terminal repeat of adeno-associated virus 
                     serotype 2"
     repeat_region   7642..7771
                     /label=AAV2 ITR
     rep_origin      7857..8312
                     /direction=RIGHT
                     /label=f1 ori
                     /note="f1 bacteriophage origin of replication; arrow 
                     indicates direction of (+) strand synthesis"
     primer_bind     complement(7944..7963)
                     /label=F1ori-R
                     /note="F1 origin, reverse primer"
     primer_bind     8154..8175
                     /label=F1ori-F
                     /note="F1 origin, forward primer"
     primer_bind     complement(8329..8348)
                     /label=pRS-marker
                     /note="pRS vectors, use to sequence yeast selectable 
                     marker"
     primer_bind     8448..8470
                     /label=pGEX 3'
                     /note="pGEX vectors, reverse primer"
     primer_bind     complement(8508..8526)
                     /label=pBRforEco
                     /note="pBR322 vectors, upsteam of EcoRI site, forward 
                     primer"
     promoter        8594..8698
                     /gene="bla"
                     /label=AmpR promoter
     CDS             join(8699..8767,8768..9559)
                     /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(8917..8936)
                     /label=Amp-R
                     /note="Ampicillin resistance gene, reverse primer"
ORIGIN
        1 gatcttcttg agatcctttt tttctgcgcg taatctgctg cttgcaaaca aaaaaaccac
       61 cgctaccagc ggtggtttgt ttgccggatc aagagctacc aactcttttt ccgaaggtaa
      121 ctggcttcag cagagcgcag ataccaaata ctgtccttct agtgtagccg tagttaggcc
      181 accacttcaa gaactctgta gcaccgccta catacctcgc tctgctaatc ctgttaccag
      241 tggctgctgc cagtggcgat aagtcgtgtc ttaccgggtt ggactcaaga cgatagttac
      301 cggataaggc gcagcggtcg ggctgaacgg ggggttcgtg cacacagccc agcttggagc
      361 gaacgaccta caccgaactg agatacctac agcgtgagct atgagaaagc gccacgcttc
      421 ccgaagggag aaaggcggac aggtatccgg taagcggcag ggtcggaaca ggagagcgca
      481 cgagggagct tccaggggga aacgcctggt atctttatag tcctgtcggg tttcgccacc
      541 tctgacttga gcgtcgattt ttgtgatgct cgtcaggggg gcggagccta tggaaaaacg
      601 ccagcaacgc ggccttttta cggttcctgg ccttttgctg gccttttgct cacatgtgag
      661 ggcctatttc ccatgattcc ttcatatttg catatacgat acaaggctgt tagagagata
      721 attggaatta atttgactgt aaacacaaag atattagtac aaaatacgtg acgtagaaag
      781 taataatttc ttgggtagtt tgcagtttta aaattatgtt ttaaaatgga ctatcatatg
      841 cttaccgtaa cttgaaagta tttcgatttc ttggctttat atatcttgtg gaaaggacga
      901 aacaccgctg caggatgatt gccgccggtt ttagagctag aaatagcaag ttaaaataag
      961 gctagtccgt tatcaacttg aaaaagtggc accgagtcgg tgcttttttg ttttagagct
     1021 agaaatagca agttaaaata aggctagtcc gtttttagcg cgtgcgccaa ttctgcagac
     1081 aaatggctct agagagggcc tatttcccat gattccttca tatttgcata tacgatacaa
     1141 ggctgttaga gagataattg gaattaattt gactgtaaac acaaagatat tagtacaaaa
     1201 tacgtgacgt agaaagtaat aatttcttgg gtagtttgca gttttaaaat tatgttttaa
     1261 aatggactat catatgctta ccgtaacttg aaagtatttc gatttcttgg ctttatatat
     1321 cttgtggaaa ggacgaaaca ccgtctgacg gcaacttcaa ctggttttag agctagaaat
     1381 agcaagttaa aataaggcta gtccgttatc aacttgaaaa agtggcaccg agtcggtgct
     1441 tttttgtttt agagctagaa atagcaagtt aaaataaggc tagtccgttt ttagcgcgtg
     1501 cgccaattct gcagacaaat ggcggtaccc gttacataac ttacggtaaa tggcccgcct
     1561 ggctgaccgc ccaacgaccc ccgcccattg acgtcaatag taacgccaat agggactttc
     1621 cattgacgtc aatgggtgga gtatttacgg taaactgccc acttggcagt acatcaagtg
     1681 tatcatatgc caagtacgcc ccctattgac gtcaatgacg gtaaatggcc cgcctggcat
     1741 tgtgcccagt acatgacctt atgggacttt cctacttggc agtacatcta cgtattagtc
     1801 atcgctatta ccatggtcga ggtgagcccc acgttctgct tcactctccc catctccccc
     1861 ccctccccac ccccaatttt gtatttattt attttttaat tattttgtgc agcgatgggg
     1921 gcgggggggg ggggggggcg cgcgccaggc ggggcggggc ggggcgaggg gcggggcggg
     1981 gcgaggcgga gaggtgcggc ggcagccaat cagagcggcg cgctccgaaa gtttcctttt
     2041 atggcgaggc ggcggcggcg gcggccctat aaaaagcgaa gcgcgcggcg ggcgggagtc
     2101 gctgcgacgc tgccttcgcc ccgtgccccg ctccgccgcc gcctcgcgcc gcccgccccg
     2161 gctctgactg accgcgttac tcccacaggt gagcgggcgg gacggccctt ctcctccggg
     2221 ctgtaattag ctgagcaaga ggtaagggtt taagggatgg ttggttggtg gggtattaat
     2281 gtttaattac ctggagcacc tgcctgaaat cacttttttt caggttggac cggtgccacc
     2341 atggactata aggaccacga cggagactac aaggatcatg atattgatta caaagacgat
     2401 gacgataaga tggccccaaa gaagaagcgg aaggtcggta tccacggagt cccagcagcc
     2461 gacaagaagt acagcatcgg cctggacatc ggcaccaact ctgtgggctg ggccgtgatc
     2521 accgacgagt acaaggtgcc cagcaagaaa ttcaaggtgc tgggcaacac cgaccggcac
     2581 agcatcaaga agaacctgat cggagccctg ctgttcgaca gcggcgaaac agccgaggcc
     2641 acccggctga agagaaccgc cagaagaaga tacaccagac ggaagaaccg gatctgctat
     2701 ctgcaagaga tcttcagcaa cgagatggcc aaggtggacg acagcttctt ccacagactg
     2761 gaagagtcct tcctggtgga agaggataag aagcacgagc ggcaccccat cttcggcaac
     2821 atcgtggacg aggtggccta ccacgagaag taccccacca tctaccacct gagaaagaaa
     2881 ctggtggaca gcaccgacaa ggccgacctg cggctgatct atctggccct ggcccacatg
     2941 atcaagttcc ggggccactt cctgatcgag ggcgacctga accccgacaa cagcgacgtg
     3001 gacaagctgt tcatccagct ggtgcagacc tacaaccagc tgttcgagga aaaccccatc
     3061 aacgccagcg gcgtggacgc caaggccatc ctgtctgcca gactgagcaa gagcagacgg
     3121 ctggaaaatc tgatcgccca gctgcccggc gagaagaaga atggcctgtt cggcaacctg
     3181 attgccctga gcctgggcct gacccccaac ttcaagagca acttcgacct ggccgaggat
     3241 gccaaactgc agctgagcaa ggacacctac gacgacgacc tggacaacct gctggcccag
     3301 atcggcgacc agtacgccga cctgtttctg gccgccaaga acctgtccga cgccatcctg
     3361 ctgagcgaca tcctgagagt gaacaccgag atcaccaagg cccccctgag cgcctctatg
     3421 atcaagagat acgacgagca ccaccaggac ctgaccctgc tgaaagctct cgtgcggcag
     3481 cagctgcctg agaagtacaa agagattttc ttcgaccaga gcaagaacgg ctacgccggc
     3541 tacattgacg gcggagccag ccaggaagag ttctacaagt tcatcaagcc catcctggaa
     3601 aagatggacg gcaccgagga actgctcgtg aagctgaaca gagaggacct gctgcggaag
     3661 cagcggacct tcgacaacgg cagcatcccc caccagatcc acctgggaga gctgcacgcc
     3721 attctgcggc ggcaggaaga tttttaccca ttcctgaagg acaaccggga aaagatcgag
     3781 aagatcctga ccttccgcat cccctactac gtgggccctc tggccagggg aaacagcaga
     3841 ttcgcctgga tgaccagaaa gagcgaggaa accatcaccc cctggaactt cgaggaagtg
     3901 gtggacaagg gcgcttccgc ccagagcttc atcgagcgga tgaccaactt cgataagaac
     3961 ctgcccaacg agaaggtgct gcccaagcac agcctgctgt acgagtactt caccgtgtat
     4021 aacgagctga ccaaagtgaa atacgtgacc gagggaatga gaaagcccgc cttcctgagc
     4081 ggcgagcaga aaaaggccat cgtggacctg ctgttcaaga ccaaccggaa agtgaccgtg
     4141 aagcagctga aagaggacta cttcaagaaa atcgagtgct tcgactccgt ggaaatctcc
     4201 ggcgtggaag atcggttcaa cgcctccctg ggcacatacc acgatctgct gaaaattatc
     4261 aaggacaagg acttcctgga caatgaggaa aacgaggaca ttctggaaga tatcgtgctg
     4321 accctgacac tgtttgagga cagagagatg atcgaggaac ggctgaaaac ctatgcccac
     4381 ctgttcgacg acaaagtgat gaagcagctg aagcggcgga gatacaccgg ctggggcagg
     4441 ctgagccgga agctgatcaa cggcatccgg gacaagcagt ccggcaagac aatcctggat
     4501 ttcctgaagt ccgacggctt cgccaacaga aacttcatgc agctgatcca cgacgacagc
     4561 ctgaccttta aagaggacat ccagaaagcc caggtgtccg gccagggcga tagcctgcac
     4621 gagcacattg ccaatctggc cggcagcccc gccattaaga agggcatcct gcagacagtg
     4681 aaggtggtgg acgagctcgt gaaagtgatg ggccggcaca agcccgagaa catcgtgatc
     4741 gaaatggcca gagagaacca gaccacccag aagggacaga agaacagccg cgagagaatg
     4801 aagcggatcg aagagggcat caaagagctg ggcagccaga tcctgaaaga acaccccgtg
     4861 gaaaacaccc agctgcagaa cgagaagctg tacctgtact acctgcagaa tgggcgggat
     4921 atgtacgtgg accaggaact ggacatcaac cggctgtccg actacgatgt ggaccatatc
     4981 gtgcctcaga gctttctgaa ggacgactcc atcgacaaca aggtgctgac cagaagcgac
     5041 aagaaccggg gcaagagcga caacgtgccc tccgaagagg tcgtgaagaa gatgaagaac
     5101 tactggcggc agctgctgaa cgccaagctg attacccaga gaaagttcga caatctgacc
     5161 aaggccgaga gaggcggcct gagcgaactg gataaggccg gcttcatcaa gagacagctg
     5221 gtggaaaccc ggcagatcac aaagcacgtg gcacagatcc tggactcccg gatgaacact
     5281 aagtacgacg agaatgacaa gctgatccgg gaagtgaaag tgatcaccct gaagtccaag
     5341 ctggtgtccg atttccggaa ggatttccag ttttacaaag tgcgcgagat caacaactac
     5401 caccacgccc acgacgccta cctgaacgcc gtcgtgggaa ccgccctgat caaaaagtac
     5461 cctaagctgg aaagcgagtt cgtgtacggc gactacaagg tgtacgacgt gcggaagatg
     5521 atcgccaaga gcgagcagga aatcggcaag gctaccgcca agtacttctt ctacagcaac
     5581 atcatgaact ttttcaagac cgagattacc ctggccaacg gcgagatccg gaagcggcct
     5641 ctgatcgaga caaacggcga aaccggggag atcgtgtggg ataagggccg ggattttgcc
     5701 accgtgcgga aagtgctgag catgccccaa gtgaatatcg tgaaaaagac cgaggtgcag
     5761 acaggcggct tcagcaaaga gtctatcctg cccaagagga acagcgataa gctgatcgcc
     5821 agaaagaagg actgggaccc taagaagtac ggcggcttcg acagccccac cgtggcctat
     5881 tctgtgctgg tggtggccaa agtggaaaag ggcaagtcca agaaactgaa gagtgtgaaa
     5941 gagctgctgg ggatcaccat catggaaaga agcagcttcg agaagaatcc catcgacttt
     6001 ctggaagcca agggctacaa agaagtgaaa aaggacctga tcatcaagct gcctaagtac
     6061 tccctgttcg agctggaaaa cggccggaag agaatgctgg cctctgccgg cgaactgcag
     6121 aagggaaacg aactggccct gccctccaaa tatgtgaact tcctgtacct ggccagccac
     6181 tatgagaagc tgaagggctc ccccgaggat aatgagcaga aacagctgtt tgtggaacag
     6241 cacaagcact acctggacga gatcatcgag cagatcagcg agttctccaa gagagtgatc
     6301 ctggccgacg ctaatctgga caaagtgctg tccgcctaca acaagcaccg ggataagccc
     6361 atcagagagc aggccgagaa tatcatccac ctgtttaccc tgaccaatct gggagcccct
     6421 gccgccttca agtactttga caccaccatc gaccggaaga ggtacaccag caccaaagag
     6481 gtgctggacg ccaccctgat ccaccagagc atcaccggcc tgtacgagac acggatcgac
     6541 ctgtctcagc tgggaggcga caaaaggccg gcggccacga aaaaggccgg ccaggcaaaa
     6601 aagaaaaagg aattcggcag tggagagggc agaggaagtc tgctaacatg cggtgacgtc
     6661 gaggagaatc ctggcccagt gagcaagggc gaggagctgt tcaccggggt ggtgcccatc
     6721 ctggtcgagc tggacggcga cgtaaacggc cacaagttca gcgtgtccgg cgagggcgag
     6781 ggcgatgcca cctacggcaa gctgaccctg aagttcatct gcaccaccgg caagctgccc
     6841 gtgccctggc ccaccctcgt gaccaccctg acctacggcg tgcagtgctt cagccgctac
     6901 cccgaccaca tgaagcagca cgacttcttc aagtccgcca tgcccgaagg ctacgtccag
     6961 gagcgcacca tcttcttcaa ggacgacggc aactacaaga cccgcgccga ggtgaagttc
     7021 gagggcgaca ccctggtgaa ccgcatcgag ctgaagggca tcgacttcaa ggaggacggc
     7081 aacatcctgg ggcacaagct ggagtacaac tacaacagcc acaacgtcta tatcatggcc
     7141 gacaagcaga agaacggcat caaggtgaac ttcaagatcc gccacaacat cgaggacggc
     7201 agcgtgcagc tcgccgacca ctaccagcag aacaccccca tcggcgacgg ccccgtgctg
     7261 ctgcccgaca accactacct gagcacccag tccgccctga gcaaagaccc caacgagaag
     7321 cgcgatcaca tggtcctgct ggagttcgtg accgccgccg ggatcactct cggcatggac
     7381 gagctgtaca aggaattcta actagagctc gctgatcagc ctcgactgtg ccttctagtt
     7441 gccagccatc tgttgtttgc ccctcccccg tgccttcctt gaccctggaa ggtgccactc
     7501 ccactgtcct ttcctaataa aatgaggaaa ttgcatcgca ttgtctgagt aggtgtcatt
     7561 ctattctggg gggtggggtg gggcaggaca gcaaggggga ggattgggaa gagaatagca
     7621 ggcatgctgg ggagcggccg caggaacccc tagtgatgga gttggccact ccctctctgc
     7681 gcgctcgctc gctcactgag gccgggcgac caaaggtcgc ccgacgcccg ggctttgccc
     7741 gggcggcctc agtgagcgag cgagcgcgca gctgcctgca ggggcgcctg atgcggtatt
     7801 ttctccttac gcatctgtgc ggtatttcac accgcatacg tcaaagcaac catagtacgc
     7861 gccctgtagc ggcgcattaa gcgcggcggg tgtggtggtt acgcgcagcg tgaccgctac
     7921 acttgccagc gccctagcgc ccgctccttt cgctttcttc ccttcctttc tcgccacgtt
     7981 cgccggcttt ccccgtcaag ctctaaatcg ggggctccct ttagggttcc gatttagtgc
     8041 tttacggcac ctcgacccca aaaaacttga tttgggtgat ggttcacgta gtgggccatc
     8101 gccctgatag acggtttttc gccctttgac gttggagtcc acgttcttta atagtggact
     8161 cttgttccaa actggaacaa cactcaaccc tatctcgggc tattcttttg atttataagg
     8221 gattttgccg atttcggcct attggttaaa aaatgagctg atttaacaaa aatttaacgc
     8281 gaattttaac aaaatattaa cgtttacaat tttatggtgc actctcagta caatctgctc
     8341 tgatgccgca tagttaagcc agccccgaca cccgccaaca cccgctgacg cgccctgacg
     8401 ggcttgtctg ctcccggcat ccgcttacag acaagctgtg accgtctccg ggagctgcat
     8461 gtgtcagagg ttttcaccgt catcaccgaa acgcgcgaga cgaaagggcc tcgtgatacg
     8521 cctattttta taggttaatg tcatgataat aatggtttct tagacgtcag gtggcacttt
     8581 tcggggaaat gtgcgcggaa cccctatttg tttatttttc taaatacatt caaatatgta
     8641 tccgctcatg agacaataac cctgataaat gcttcaataa tattgaaaaa ggaagagtat
     8701 gagtattcaa catttccgtg tcgcccttat tccctttttt gcggcatttt gccttcctgt
     8761 ttttgctcac ccagaaacgc tggtgaaagt aaaagatgct gaagatcagt tgggtgcacg
     8821 agtgggttac atcgaactgg atctcaacag cggtaagatc cttgagagtt ttcgccccga
     8881 agaacgtttt ccaatgatga gcacttttaa agttctgcta tgtggcgcgg tattatcccg
     8941 tattgacgcc gggcaagagc aactcggtcg ccgcatacac tattctcaga atgacttggt
     9001 tgagtactca ccagtcacag aaaagcatct tacggatggc atgacagtaa gagaattatg
     9061 cagtgctgcc ataaccatga gtgataacac tgcggccaac ttacttctga caacgatcgg
     9121 aggaccgaag gagctaaccg cttttttgca caacatgggg gatcatgtaa ctcgccttga
     9181 tcgttgggaa ccggagctga atgaagccat accaaacgac gagcgtgaca ccacgatgcc
     9241 tgtagcaatg gcaacaacgt tgcgcaaact attaactggc gaactactta ctctagcttc
     9301 ccggcaacaa ttaatagact ggatggaggc ggataaagtt gcaggaccac ttctgcgctc
     9361 ggcccttccg gctggctggt ttattgctga taaatctgga gccggtgagc gtggaagccg
     9421 cggtatcatt gcagcactgg ggccagatgg taagccctcc cgtatcgtag ttatctacac
     9481 gacggggagt caggcaacta tggatgaacg aaatagacag atcgctgaga taggtgcctc
     9541 actgattaag cattggtaac tgtcagacca agtttactca tatatacttt agattgattt
     9601 aaaacttcat ttttaattta aaaggatcta ggtgaagatc ctttttgata atctcatgac
     9661 caaaatccct taacgtgagt tttcgttcca ctgagcgtca gaccccgtag aaaagatcaa
     9721 ag
//
