LOCUS       sequence-487610-        9452 bp    DNA     circular SYN 01-OCT-2025
DEFINITION  CBh-SpCas9-2A-Puro, and 2X hU6-sgRNA (Sp) with BbsI golden gate 
            cloning backbone dual gRNAs. sgRNA uses 3TC scaffold for increased 
            sgRNA expression. .
ACCESSION   .
VERSION     .
KEYWORDS    .
SOURCE      synthetic DNA construct
  ORGANISM  synthetic DNA construct
REFERENCE   1  (bases 1 to 9452)
  AUTHORS   Chey YCJ, Gierus L, Lushington C, Arudkumar JC, B Geiger A, Staker 
            LG, Robertson LJ, Pfitzner C, Kennedy JG, Lee RHB, Godahewa GI, 
            Adikusuma F, Thomas PQ
  TITLE     Optimal SpCas9- and SaCas9-mediated gene editing by enhancing gRNA 
            transcript levels through scaffold poly-T tract reduction.
  JOURNAL   BMC Genomics. 2025 Feb 12;26(1):138. doi: 
            10.1186/s12864-025-11317-2.
  PUBMED    39939860
REFERENCE   2  (bases 1 to 9452)
  AUTHORS   .
  TITLE     Direct Submission
  JOURNAL   Exported Oct 1, 2025 from SnapGene Server 8.0.1
            https://www.snapgene.com
COMMENT     SGRef: number: 1; type: "Journal Article"; journalName: "BMC 
            Genomics."; date: "2025-02-12"; volume: "26(1):138. doi"; pages: " 
            10.1186/s12864-025-11317-2"
COMMENT     Sequence Label: PDG459 V3
FEATURES             Location/Qualifiers
     source          1..9452
                     /mol_type="other DNA"
                     /organism="synthetic DNA construct"
     rep_origin      1..589
                     /direction=RIGHT
                     /label=ori
                     /note="high-copy-number ColE1/pMB1/pBR322/pUC origin of 
                     replication"
     primer_bind     490..509
                     /label=pBR322ori-F
                     /note="pBR322 origin, forward primer"
     promoter        651..899
                     /label=U6 promoter
                     /note="RNA polymerase III promoter for human U6 snRNA 
                     (Domitrovich & Kunkel, 2003)"
     misc_RNA        918..993
                     /label=gRNA scaffold
                     /note="guide RNA scaffold for the Streptococcus pyogenes 
                     CRISPR/Cas9 system"
     enhancer        1090..1375
                     /label=CMV enhancer
                     /note="human cytomegalovirus immediate early enhancer; 
                     contains an 18-bp deletion relative to the standard CMV 
                     enhancer"
     promoter        1377..1654
                     /label=chicken beta-actin promoter
     intron          1655..1882
                     /label=hybrid intron
                     /note="hybrid between chicken beta-actin (CBA) and minute 
                     virus of mice (MMV) introns (Gray et al., 2011)"
     regulatory      1894..1903
                     /label=Kozak sequence
                     /note="vertebrate consensus sequence for strong initiation 
                     of translation (Kozak, 1987)"
                     /regulatory_class="other"
     CDS             1903..1968
                     /codon_start=1
                     /product="three tandem FLAG(R) epitope tags, followed by an
                     enterokinase cleavage site"
                     /label=3xFLAG
                     /translation="DYKDHDGDYKDHDIDYKDDDDK"
     CDS             1975..1995
                     /codon_start=1
                     /product="nuclear localization signal of SV40 (simian virus
                     40) large T antigen"
                     /label=SV40 NLS
                     /translation="PKKKRKV"
     CDS             2020..6120
                     /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             2020..6120
                     /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             6121..6168
                     /codon_start=1
                     /product="bipartite nuclear localization signal from 
                     nucleoplasmin"
                     /label=nucleoplasmin NLS
                     /translation="KRPAATKKAGQAKKKK"
     CDS             6184..6237
                     /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             6238..6837
                     /codon_start=1
                     /gene="pac from Streptomyces alboniger"
                     /product="puromycin N-acetyltransferase"
                     /label=PuroR
                     /note="confers resistance to puromycin"
                     /translation="MTEYKPTVRLATRDDVPRAVRTLAAAFADYPATRHTVDPDRHIER
                     VTELQELFLTRVGLDIGKVWVADDGAAVAVWTTPESVEAGAVFAEIGPRMAELSGSRLA
                     AQQQMEGLLAPHRPKEPAWFLATVGVSPDHQGKGLGSAVVLPGVEAAERAGVPAFLETS
                     APRNLPFYERLGFTVTADVEVPEGPRTWCMTRKPGA"
     primer_bind     complement(6238..6257)
                     /label=Puro-R
                     /note="Puromycin resistance gene, reverse primer. Also 
                     called puro-variant-R"
     primer_bind     6734..6754
                     /label=Puro-F
                     /note="Puromycin resistance gene, forward primer"
     primer_bind     complement(6865..6882)
                     /label=BGH-rev
                     /note="Bovine growth hormone terminator, reverse primer. 
                     Also called BGH reverse"
     polyA_signal    6871..7078
                     /label=bGH poly(A) signal
                     /note="bovine growth hormone polyadenylation signal"
     misc_RNA        complement(7156..7231)
                     /label=gRNA scaffold
                     /note="guide RNA scaffold for the Streptococcus pyogenes 
                     CRISPR/Cas9 system"
     promoter        complement(7249..7497)
                     /label=U6 promoter
                     /note="RNA polymerase III promoter for human U6 snRNA 
                     (Domitrovich & Kunkel, 2003)"
     rep_origin      7580..8035
                     /direction=RIGHT
                     /label=f1 ori
                     /note="f1 bacteriophage origin of replication; arrow 
                     indicates direction of (+) strand synthesis"
     primer_bind     complement(7667..7686)
                     /label=F1ori-R
                     /note="F1 origin, reverse primer"
     primer_bind     7877..7898
                     /label=F1ori-F
                     /note="F1 origin, forward primer"
     primer_bind     complement(8052..8071)
                     /label=pRS-marker
                     /note="pRS vectors, use to sequence yeast selectable 
                     marker"
     primer_bind     8171..8193
                     /label=pGEX 3'
                     /note="pGEX vectors, reverse primer"
     primer_bind     complement(8231..8249)
                     /label=pBRforEco
                     /note="pBR322 vectors, upsteam of EcoRI site, forward 
                     primer"
     promoter        8317..8421
                     /gene="bla"
                     /label=AmpR promoter
     CDS             join(8422..8490,8491..9282)
                     /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(8640..8659)
                     /label=Amp-R
                     /note="Ampicillin resistance gene, reverse primer"
ORIGIN
        1 ttgagatcct ttttttctgc gcgtaatctg ctgcttgcaa acaaaaaaac caccgctacc
       61 agcggtggtt tgtttgccgg atcaagagct accaactctt tttccgaagg taactggctt
      121 cagcagagcg cagataccaa atactgttct tctagtgtag ccgtagttag gccaccactt
      181 caagaactct gtagcaccgc ctacatacct cgctctgcta atcctgttac cagtggctgc
      241 tgccagtggc gataagtcgt gtcttaccgg gttggactca agacgatagt taccggataa
      301 ggcgcagcgg tcgggctgaa cggggggttc gtgcacacag cccagcttgg agcgaacgac
      361 ctacaccgaa ctgagatacc tacagcgtga gctatgagaa agcgccacgc ttcccgaagg
      421 gagaaaggcg gacaggtatc cggtaagcgg cagggtcgga acaggagagc gcacgaggga
      481 gcttccaggg ggaaacgcct ggtatcttta tagtcctgtc gggtttcgcc acctctgact
      541 tgagcgtcga tttttgtgat gctcgtcagg ggggcggagc ctatggaaaa acgccagcaa
      601 cgcggccttt ttacggttcc tggccttttg ctggcctttt gctcacatgt gagggcctat
      661 ttcccatgat tccttcatat ttgcatatac gatacaaggc tgttagagag ataattggaa
      721 ttaatttgac tgtaaacaca aagatattag tacaaaatac gtgacgtaga aagtaataat
      781 ttcttgggta gtttgcagtt ttaaaattat gttttaaaat ggactatcat atgcttaccg
      841 taacttgaaa gtatttcgat ttcttggctt tatatatctt gtggaaagga cgaaacaccg
      901 ggtcttcgag aagacctgtt tcagagctag aaatagcaag ttgaaataag gctagtccgt
      961 tatcaacttg aaaaagtggc accgagtcgg tgcttttttg ttttagagct agaaatagca
     1021 agttaaaata aggctagtcc gtttttagcg cgtgcgccaa ttctgcagac aaatggctct
     1081 agaggtaccc gttacataac ttacggtaaa tggcccgcct ggctgaccgc ccaacgaccc
     1141 ccgcccattg acgtcaatag taacgccaat agggactttc cattgacgtc aatgggtgga
     1201 gtatttacgg taaactgccc acttggcagt acatcaagtg tatcatatgc caagtacgcc
     1261 ccctattgac gtcaatgacg gtaaatggcc cgcctggcat tgtgcccagt acatgacctt
     1321 atgggacttt cctacttggc agtacatcta cgtattagtc atcgctatta ccatggtcga
     1381 ggtgagcccc acgttctgct tcactctccc catctccccc ccctccccac ccccaatttt
     1441 gtatttattt attttttaat tattttgtgc agcgatgggg gcgggggggg ggggggggcg
     1501 cgcgccaggc ggggcggggc ggggcgaggg gcggggcggg gcgaggcgga gaggtgcggc
     1561 ggcagccaat cagagcggcg cgctccgaaa gtttcctttt atggcgaggc ggcggcggcg
     1621 gcggccctat aaaaagcgaa gcgcgcggcg ggcgggagtc gctgcgcgct gccttcgccc
     1681 cgtgccccgc tccgccgccg cctcgcgccg cccgccccgg ctctgactga ccgcgttact
     1741 cccacaggtg agcgggcggg acggcccttc tcctccgggc tgtaattagc tgagcaagag
     1801 gtaagggttt aagggatggt tggttggtgg ggtattaatg tttaattacc tggagcacct
     1861 gcctgaaatc actttttttc aggttggacc ggtgccacca tggactataa ggaccacgac
     1921 ggagactaca aggatcatga tattgattac aaagacgatg acgataagat ggccccaaag
     1981 aagaagcgga aggtcggtat ccacggagtc ccagcagccg acaagaagta cagcatcggc
     2041 ctggacatcg gcaccaactc tgtgggctgg gccgtgatca ccgacgagta caaggtgccc
     2101 agcaagaaat tcaaggtgct gggcaacacc gaccggcaca gcatcaagaa gaacctgatc
     2161 ggagccctgc tgttcgacag cggcgaaaca gccgaggcca cccggctgaa gagaaccgcc
     2221 agaagaagat acaccagacg gaagaaccgg atctgctatc tgcaagagat cttcagcaac
     2281 gagatggcca aggtggacga cagcttcttc cacagactgg aagagtcctt cctggtggaa
     2341 gaggataaga agcacgagcg gcaccccatc ttcggcaaca tcgtggacga ggtggcctac
     2401 cacgagaagt accccaccat ctaccacctg agaaagaaac tggtggacag caccgacaag
     2461 gccgacctgc ggctgatcta tctggccctg gcccacatga tcaagttccg gggccacttc
     2521 ctgatcgagg gcgacctgaa ccccgacaac agcgacgtgg acaagctgtt catccagctg
     2581 gtgcagacct acaaccagct gttcgaggaa aaccccatca acgccagcgg cgtggacgcc
     2641 aaggccatcc tgtctgccag actgagcaag agcagacggc tggaaaatct gatcgcccag
     2701 ctgcccggcg agaagaagaa tggcctgttc ggaaacctga ttgccctgag cctgggcctg
     2761 acccccaact tcaagagcaa cttcgacctg gccgaggatg ccaaactgca gctgagcaag
     2821 gacacctacg acgacgacct ggacaacctg ctggcccaga tcggcgacca gtacgccgac
     2881 ctgtttctgg ccgccaagaa cctgtccgac gccatcctgc tgagcgacat cctgagagtg
     2941 aacaccgaga tcaccaaggc ccccctgagc gcctctatga tcaagagata cgacgagcac
     3001 caccaggacc tgaccctgct gaaagctctc gtgcggcagc agctgcctga gaagtacaaa
     3061 gagattttct tcgaccagag caagaacggc tacgccggct acattgacgg cggagccagc
     3121 caggaagagt tctacaagtt catcaagccc atcctggaaa agatggacgg caccgaggaa
     3181 ctgctcgtga agctgaacag agaggacctg ctgcggaagc agcggacctt cgacaacggc
     3241 agcatccccc accagatcca cctgggagag ctgcacgcca ttctgcggcg gcaggaagat
     3301 ttttacccat tcctgaagga caaccgggaa aagatcgaga agatcctgac cttccgcatc
     3361 ccctactacg tgggccctct ggccagggga aacagcagat tcgcctggat gaccagaaag
     3421 agcgaggaaa ccatcacccc ctggaacttc gaggaagtgg tggacaaggg cgcttccgcc
     3481 cagagcttca tcgagcggat gaccaacttc gataagaacc tgcccaacga gaaggtgctg
     3541 cccaagcaca gcctgctgta cgagtacttc accgtgtata acgagctgac caaagtgaaa
     3601 tacgtgaccg agggaatgag aaagcccgcc ttcctgagcg gcgagcagaa aaaggccatc
     3661 gtggacctgc tgttcaagac caaccggaaa gtgaccgtga agcagctgaa agaggactac
     3721 ttcaagaaaa tcgagtgctt cgactccgtg gaaatctccg gcgtggaaga tcggttcaac
     3781 gcctccctgg gcacatacca cgatctgctg aaaattatca aggacaagga cttcctggac
     3841 aatgaggaaa acgaggacat tctggaagat atcgtgctga ccctgacact gtttgaggac
     3901 agagagatga tcgaggaacg gctgaaaacc tatgcccacc tgttcgacga caaagtgatg
     3961 aagcagctga agcggcggag atacaccggc tggggcaggc tgagccggaa gctgatcaac
     4021 ggcatccggg acaagcagtc cggcaagaca atcctggatt tcctgaagtc cgacggcttc
     4081 gccaacagaa acttcatgca gctgatccac gacgacagcc tgacctttaa agaggacatc
     4141 cagaaagccc aggtgtccgg ccagggcgat agcctgcacg agcacattgc caatctggcc
     4201 ggcagccccg ccattaagaa gggcatcctg cagacagtga aggtggtgga cgagctcgtg
     4261 aaagtgatgg gccggcacaa gcccgagaac atcgtgatcg aaatggccag agagaaccag
     4321 accacccaga agggacagaa gaacagccgc gagagaatga agcggatcga agagggcatc
     4381 aaagagctgg gcagccagat cctgaaagaa caccccgtgg aaaacaccca gctgcagaac
     4441 gagaagctgt acctgtacta cctgcagaat gggcgggata tgtacgtgga ccaggaactg
     4501 gacatcaacc ggctgtccga ctacgatgtg gaccatatcg tgcctcagag ctttctgaag
     4561 gacgactcca tcgacaacaa ggtgctgacc agaagcgaca agaaccgggg caagagcgac
     4621 aacgtgccct ccgaagaggt cgtgaagaag atgaagaact actggcggca gctgctgaac
     4681 gccaagctga ttacccagag aaagttcgac aatctgacca aggccgagag aggcggcctg
     4741 agcgaactgg ataaggccgg cttcatcaag agacagctgg tggaaacccg gcagatcaca
     4801 aagcacgtgg cacagatcct ggactcccgg atgaacacta agtacgacga gaatgacaag
     4861 ctgatccggg aagtgaaagt gatcaccctg aagtccaagc tggtgtccga tttccggaag
     4921 gatttccagt tttacaaagt gcgcgagatc aacaactacc accacgccca cgacgcctac
     4981 ctgaacgccg tcgtgggaac cgccctgatc aaaaagtacc ctaagctgga aagcgagttc
     5041 gtgtacggcg actacaaggt gtacgacgtg cggaagatga tcgccaagag cgagcaggaa
     5101 atcggcaagg ctaccgccaa gtacttcttc tacagcaaca tcatgaactt tttcaagacc
     5161 gagattaccc tggccaacgg cgagatccgg aagcggcctc tgatcgagac aaacggcgaa
     5221 accggggaga tcgtgtggga taagggccgg gattttgcca ccgtgcggaa agtgctgagc
     5281 atgccccaag tgaatatcgt gaaaaagacc gaggtgcaga caggcggctt cagcaaagag
     5341 tctatcctgc ccaagaggaa cagcgataag ctgatcgcca gaaagaagga ctgggaccct
     5401 aagaagtacg gcggcttcga cagccccacc gtggcctatt ctgtgctggt ggtggccaaa
     5461 gtggaaaagg gcaagtccaa gaaactgaag agtgtgaaag agctgctggg gatcaccatc
     5521 atggaaagaa gcagcttcga gaagaatccc atcgactttc tggaagccaa gggctacaaa
     5581 gaagtgaaaa aggacctgat catcaagctg cctaagtact ccctgttcga gctggaaaac
     5641 ggccggaaga gaatgctggc ctctgccggc gaactgcaga agggaaacga actggccctg
     5701 ccctccaaat atgtgaactt cctgtacctg gccagccact atgagaagct gaagggctcc
     5761 cccgaggata atgagcagaa acagctgttt gtggaacagc acaagcacta cctggacgag
     5821 atcatcgagc agatcagcga gttctccaag agagtgatcc tggccgacgc taatctggac
     5881 aaagtgctgt ccgcctacaa caagcaccgg gataagccca tcagagagca ggccgagaat
     5941 atcatccacc tgtttaccct gaccaatctg ggagcccctg ccgccttcaa gtactttgac
     6001 accaccatcg accggaagag gtacaccagc accaaagagg tgctggacgc caccctgatc
     6061 caccagagca tcaccggcct gtacgagaca cggatcgacc tgtctcagct gggaggcgac
     6121 aaaaggccgg cggccacgaa aaaggccggc caggcaaaaa agaaaaagga attcggcagt
     6181 ggagagggca gaggaagtct gctaacatgc ggtgacgtcg aggagaatcc tggcccaatg
     6241 accgagtaca agcccacggt gcgcctcgcc acccgcgacg acgtccccag ggccgtacgc
     6301 accctcgccg ccgcgttcgc cgactacccc gccacgcgcc acaccgtcga tccggaccgc
     6361 cacatcgagc gggtcaccga gctgcaagaa ctcttcctca cgcgcgtcgg gctcgacatc
     6421 ggcaaggtgt gggtcgcgga cgacggcgcc gcggtggcgg tctggaccac gccggagagc
     6481 gtcgaagcgg gggcggtgtt cgccgagatc ggcccgcgca tggccgagtt gagcggttcc
     6541 cggctggccg cgcagcaaca gatggaaggc ctcctggcgc cgcaccggcc caaggagccc
     6601 gcgtggttcc tggccaccgt cggagtctcg cccgaccacc agggcaaggg tctgggcagc
     6661 gccgtcgtgc tccccggagt ggaggcggcc gagcgcgccg gggtgcccgc cttcctggag
     6721 acctccgcgc cccgcaacct ccccttctac gagcggctcg gcttcaccgt caccgccgac
     6781 gtcgaggtgc ccgaaggacc gcgcacctgg tgcatgaccc gcaagcccgg tgcctgagaa
     6841 ttctaactag agctcgctga tcagcctcga ctgtgccttc tagttgccag ccatctgttg
     6901 tttgcccctc ccccgtgcct tccttgaccc tggaaggtgc cactcccact gtcctttcct
     6961 aataaaatga ggaaattgca tcgcattgtc tgagtaggtg tcattctatt ctggggggtg
     7021 gggtggggca ggacagcaag ggggaggatt gggaagagaa tagcaggcat gctggggagc
     7081 ggccgcaatt ggcgcacgcg ctaaaaacgg actagcctta ttttaacttg ctatttctag
     7141 ctctaaaaca aaaaagcacc gactcggtgc cactttttca agttgataac ggactagcct
     7201 tatttcaact tgctatttct agctctgaaa cgtcttctcg aagaccccgg tgtttcgtcc
     7261 tttccacaag atatataaag ccaagaaatc gaaatacttt caagttacgg taagcatatg
     7321 atagtccatt ttaaaacata attttaaaac tgcaaactac ccaagaaatt attactttct
     7381 acgtcacgta ttttgtacta atatctttgt gtttacagtc aaattaattc caattatctc
     7441 tctaacagcc ttgtatcgta tatgcaaata tgaaggaatc atgggaaata ggccctccct
     7501 gcaggggcgc ctgatgcggt attttctcct tacgcatctg tgcggtattt cacaccgcat
     7561 acgtcaaagc aaccatagta cgcgccctgt agcggcgcat taagcgcggc gggtgtggtg
     7621 gttacgcgca gcgtgaccgc tacacttgcc agcgccttag cgcccgctcc tttcgctttc
     7681 ttcccttcct ttctcgccac gttcgccggc tttccccgtc aagctctaaa tcgggggctc
     7741 cctttagggt tccgatttag tgctttacgg cacctcgacc ccaaaaaact tgatttgggt
     7801 gatggttcac gtagtgggcc atcgccctga tagacggttt ttcgcccttt gacgttggag
     7861 tccacgttct ttaatagtgg actcttgttc caaactggaa caacactcaa ctctatctcg
     7921 ggctattctt ttgatttata agggattttg ccgatttcgg tctattggtt aaaaaatgag
     7981 ctgatttaac aaaaatttaa cgcgaatttt aacaaaatat taacgtttac aattttatgg
     8041 tgcactctca gtacaatctg ctctgatgcc gcatagttaa gccagccccg acacccgcca
     8101 acacccgctg acgcgccctg acgggcttgt ctgctcccgg catccgctta cagacaagct
     8161 gtgaccgtct ccgggagctg catgtgtcag aggttttcac cgtcatcacc gaaacgcgcg
     8221 agacgaaagg gcctcgtgat acgcctattt ttataggtta atgtcatgat aataatggtt
     8281 tcttagacgt caggtggcac ttttcgggga aatgtgcgcg gaacccctat ttgtttattt
     8341 ttctaaatac attcaaatat gtatccgctc atgagacaat aaccctgata aatgcttcaa
     8401 taatattgaa aaaggaagag tatgagtatt caacatttcc gtgtcgccct tattcccttt
     8461 tttgcggcat tttgccttcc tgtttttgct cacccagaaa cgctggtgaa agtaaaagat
     8521 gctgaagatc agttgggtgc acgagtgggt tacatcgaac tggatctcaa cagcggtaag
     8581 atccttgaga gttttcgccc cgaagaacgt tttccaatga tgagcacttt taaagttctg
     8641 ctatgtggcg cggtattatc ccgtattgac gccgggcaag agcaactcgg tcgccgcata
     8701 cactattctc agaatgactt ggttgagtac tcaccagtca cagaaaagca tcttacggat
     8761 ggcatgacag taagagaatt atgcagtgct gccataacca tgagtgataa cactgcggcc
     8821 aacttacttc tgacaacgat cggaggaccg aaggagctaa ccgctttttt gcacaacatg
     8881 ggggatcatg taactcgcct tgatcgttgg gaaccggagc tgaatgaagc cataccaaac
     8941 gacgagcgtg acaccacgat gcctgtagca atggcaacaa cgttgcgcaa actattaact
     9001 ggcgaactac ttactctagc ttcccggcaa caattaatag actggatgga ggcggataaa
     9061 gttgcaggac cacttctgcg ctcggccctt ccggctggct ggtttattgc tgataaatct
     9121 ggagccggtg agcgtggaag ccgcggtatc attgcagcac tggggccaga tggtaagccc
     9181 tcccgtatcg tagttatcta cacgacgggg agtcaggcaa ctatggatga acgaaataga
     9241 cagatcgctg agataggtgc ctcactgatt aagcattggt aactgtcaga ccaagtttac
     9301 tcatatatac tttagattga tttaaaactt catttttaat ttaaaaggat ctaggtgaag
     9361 atcctttttg ataatctcat gaccaaaatc ccttaacgtg agttttcgtt ccactgagcg
     9421 tcagaccccg tagaaaagat caaaggatct tc
//
