LOCUS       sequence-140494-        9152 bp    DNA     circular SYN 15-DEC-2025
DEFINITION  Human codon optimized plasmid to express Cas9 t2a mCherry w/o NLS 
            and a sgRNA.
ACCESSION   .
VERSION     .
KEYWORDS    .
SOURCE      synthetic DNA construct
  ORGANISM  synthetic DNA construct
REFERENCE   1  (bases 1 to 9152)
  AUTHORS   Oakes BL, Nadler DC, Flamholz A, Fellmann C, Staahl BT, Doudna JA, 
            Savage DF
  TITLE     Profiling of engineering hotspots identifies an allosteric 
            CRISPR-Cas9 switch.
  JOURNAL   Nat Biotechnol. 2016 May 2. doi: 10.1038/nbt.3528.
  PUBMED    27136077
REFERENCE   2  (bases 1 to 9152)
  AUTHORS   .
  TITLE     Direct Submission
  JOURNAL   Exported Dec 15, 2025 from SnapGene Server 8.0.1
            https://www.snapgene.com
COMMENT     SGRef: number: 1; type: "Journal Article"; journalName: "Nat 
            Biotechnol. 2016 May 2. doi: 10.1038/nbt.3528."
COMMENT     Sequence Label: pBLO1811_Cas9_noNLS_human
FEATURES             Location/Qualifiers
     source          1..9152
                     /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)"
     primer_bind     651..671
                     /label=hU6-F
                     /note="Human U6 promoter, forward primer"
     primer_bind     822..841
                     /label=LKO.1 5'
                     /note="Human U6 promoter, forward primer"
     enhancer        1100..1385
                     /label=CMV enhancer
                     /note="human cytomegalovirus immediate early enhancer; 
                     contains an 18-bp deletion relative to the standard CMV 
                     enhancer"
     promoter        1387..1664
                     /label=chicken beta-actin promoter
     intron          1665..1893
                     /label=hybrid intron
                     /note="hybrid between chicken beta-actin (CBA) and minute 
                     virus of mice (MMV) introns (Gray et al., 2011)"
     CDS             1908..1931
                     /codon_start=1
                     /product="FLAG(R) epitope tag, followed by an enterokinase 
                     cleavage site"
                     /label=FLAG
                     /translation="DYKDDDDK"
     CDS             1932..6032
                     /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             1932..6032
                     /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             6054..6107
                     /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             6108..6818
                     /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(6253..6271)
                     /label=mCherry-R
                     /note="mCherry, reverse primer"
     primer_bind     complement(6843..6860)
                     /label=BGH-rev
                     /note="Bovine growth hormone terminator, reverse primer. 
                     Also called BGH reverse"
     polyA_signal    6849..7056
                     /label=bGH poly(A) signal
                     /note="bovine growth hormone polyadenylation signal"
     repeat_region   7065..7205
                     /label=AAV2 ITR
                     /note="inverted terminal repeat of adeno-associated virus 
                     serotype 2"
     repeat_region   7065..7194
                     /label=AAV2 ITR
     rep_origin      7280..7735
                     /direction=RIGHT
                     /label=f1 ori
                     /note="f1 bacteriophage origin of replication; arrow 
                     indicates direction of (+) strand synthesis"
     primer_bind     complement(7367..7386)
                     /label=F1ori-R
                     /note="F1 origin, reverse primer"
     primer_bind     7577..7598
                     /label=F1ori-F
                     /note="F1 origin, forward primer"
     primer_bind     complement(7752..7771)
                     /label=pRS-marker
                     /note="pRS vectors, use to sequence yeast selectable 
                     marker"
     primer_bind     7871..7893
                     /label=pGEX 3'
                     /note="pGEX vectors, reverse primer"
     primer_bind     complement(7931..7949)
                     /label=pBRforEco
                     /note="pBR322 vectors, upsteam of EcoRI site, forward 
                     primer"
     promoter        8017..8121
                     /gene="bla"
                     /label=AmpR promoter
     CDS             join(8122..8190,8191..8982)
                     /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(8340..8359)
                     /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 atactgtcct 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 taagagctat gctggaaaca gcatagcaag tttaaataag
      961 gctagtccgt tatcaacttg aaaaagtggc accgagtcgg tgcttttttg ttttagagct
     1021 agaaatagca agttaaaata aggctagtcc gtttttagcg cgtgcgccaa ttctgcagac
     1081 aaatggctct agaggtaccc gttacataac ttacggtaaa tggcccgcct ggctgaccgc
     1141 ccaacgaccc ccgcccattg acgtcaatag taacgccaat agggactttc cattgacgtc
     1201 aatgggtgga gtatttacgg taaactgccc acttggcagt acatcaagtg tatcatatgc
     1261 caagtacgcc ccctattgac gtcaatgacg gtaaatggcc cgcctggcat tgtgcccagt
     1321 acatgacctt atgggacttt cctacttggc agtacatcta cgtattagtc atcgctatta
     1381 ccatggtcga ggtgagcccc acgttctgct tcactctccc catctccccc ccctccccac
     1441 ccccaatttt gtatttattt attttttaat tattttgtgc agcgatgggg gcgggggggg
     1501 ggggggggcg cgcgccaggc ggggcggggc ggggcgaggg gcggggcggg gcgaggcgga
     1561 gaggtgcggc ggcagccaat cagagcggcg cgctccgaaa gtttcctttt atggcgaggc
     1621 ggcggcggcg gcggccctat aaaaagcgaa gcgcgcggcg ggcgggagtc gctgcgacgc
     1681 tgccttcgcc ccgtgccccg ctccgccgcc gcctcgcgcc gcccgccccg gctctgactg
     1741 accgcgttac tcccacaggt gagcgggcgg gacggccctt ctcctccggg ctgtaattag
     1801 ctgagcaaga ggtaagggtt taagggatgg ttggttggtg gggtattaat gtttaattac
     1861 ctggagcacc tgcctgaaat cacttttttt caggttggac cggtatggat tacaaagacg
     1921 atgacgataa ggacaagaag tacagcatcg gcctggacat cggcaccaac tctgtgggct
     1981 gggccgtgat caccgacgag tacaaggtgc ccagcaagaa attcaaggtg ctgggcaaca
     2041 ccgaccggca cagcatcaag aagaacctga tcggagccct gctgttcgac agcggcgaaa
     2101 cagccgaggc cacccggctg aagagaaccg ccagaagaag atacaccaga cggaagaacc
     2161 ggatctgcta tctgcaagag atcttcagca acgagatggc caaggtggac gacagcttct
     2221 tccacagact ggaagagtcc ttcctggtgg aagaggataa gaagcacgag cggcacccca
     2281 tcttcggcaa catcgtggac gaggtggcct accacgagaa gtaccccacc atctaccacc
     2341 tgagaaagaa actggtggac agcaccgaca aggccgacct gcggctgatc tatctggccc
     2401 tggcccacat gatcaagttc cggggccact tcctgatcga gggcgacctg aaccccgaca
     2461 acagcgacgt ggacaagctg ttcatccagc tggtgcagac ctacaaccag ctgttcgagg
     2521 aaaaccccat caacgccagc ggcgtggacg ccaaggccat cctgtctgcc agactgagca
     2581 agagcagacg gctggaaaat ctgatcgccc agctgcccgg cgagaagaag aatggcctgt
     2641 tcggcaacct gattgccctg agcctgggcc tgacccccaa cttcaagagc aacttcgacc
     2701 tggccgagga tgccaaactg cagctgagca aggacaccta cgacgacgac ctggacaacc
     2761 tgctggccca gatcggcgac cagtacgccg acctgtttct ggccgccaag aacctgtccg
     2821 acgccatcct gctgagcgac atcctgagag tgaacaccga gatcaccaag gcccccctga
     2881 gcgcctctat gatcaagaga tacgacgagc accaccagga cctgaccctg ctgaaagctc
     2941 tcgtgcggca gcagctgcct gagaagtaca aagagatttt cttcgaccag agcaagaacg
     3001 gctacgccgg ctacattgac ggcggagcca gccaggaaga gttctacaag ttcatcaagc
     3061 ccatcctgga aaagatggac ggcaccgagg aactgctcgt gaagctgaac agagaggacc
     3121 tgctgcggaa gcagcggacc ttcgacaacg gcagcatccc ccaccagatc cacctgggag
     3181 agctgcacgc cattctgcgg cggcaggaag atttttaccc attcctgaag gacaaccggg
     3241 aaaagatcga gaagatcctg accttccgca tcccctacta cgtgggccct ctggccaggg
     3301 gaaacagcag attcgcctgg atgaccagaa agagcgagga aaccatcacc ccctggaact
     3361 tcgaggaagt ggtggacaag ggcgcttccg cccagagctt catcgagcgg atgaccaact
     3421 tcgataagaa cctgcccaac gagaaggtgc tgcccaagca cagcctgctg tacgagtact
     3481 tcaccgtgta taacgagctg accaaagtga aatacgtgac cgagggaatg agaaagcccg
     3541 ccttcctgag cggcgagcag aaaaaggcca tcgtggacct gctgttcaag accaaccgga
     3601 aagtgaccgt gaagcagctg aaagaggact acttcaagaa aatcgagtgc ttcgactccg
     3661 tggaaatctc cggcgtggaa gatcggttca acgcctccct gggcacatac cacgatctgc
     3721 tgaaaattat caaggacaag gacttcctgg acaatgagga aaacgaggac attctggaag
     3781 atatcgtgct gaccctgaca ctgtttgagg acagagagat gatcgaggaa cggctgaaaa
     3841 cctatgccca cctgttcgac gacaaagtga tgaagcagct gaagcggcgg agatacaccg
     3901 gctggggcag gctgagccgg aagctgatca acggcatccg ggacaagcag tccggcaaga
     3961 caatcctgga tttcctgaag tccgacggct tcgccaacag aaacttcatg cagctgatcc
     4021 acgacgacag cctgaccttt aaagaggaca tccagaaagc ccaggtgtcc ggccagggcg
     4081 atagcctgca cgagcacatt gccaatctgg ccggcagccc cgccattaag aagggcatcc
     4141 tgcagacagt gaaggtggtg gacgagctcg tgaaagtgat gggccggcac aagcccgaga
     4201 acatcgtgat cgaaatggcc agagagaacc agaccaccca gaagggacag aagaacagcc
     4261 gcgagagaat gaagcggatc gaagagggca tcaaagagct gggcagccag atcctgaaag
     4321 aacaccccgt ggaaaacacc cagctgcaga acgagaagct gtacctgtac tacctgcaga
     4381 atgggcggga tatgtacgtg gaccaggaac tggacatcaa ccggctgtcc gactacgatg
     4441 tggaccatat cgtgcctcag agctttctga aggacgactc catcgacaac aaggtgctga
     4501 ccagaagcga caagaaccgg ggcaagagcg acaacgtgcc ctccgaagag gtcgtgaaga
     4561 agatgaagaa ctactggcgg cagctgctga acgccaagct gattacccag agaaagttcg
     4621 acaatctgac caaggccgag agaggcggcc tgagcgaact ggataaggcc ggcttcatca
     4681 agagacagct ggtggaaacc cggcagatca caaagcacgt ggcacagatc ctggactccc
     4741 ggatgaacac taagtacgac gagaatgaca agctgatccg ggaagtgaaa gtgatcaccc
     4801 tgaagtccaa gctggtgtcc gatttccgga aggatttcca gttttacaaa gtgcgcgaga
     4861 tcaacaacta ccaccacgcc cacgacgcct acctgaacgc cgtcgtggga accgccctga
     4921 tcaaaaagta ccctaagctg gaaagcgagt tcgtgtacgg cgactacaag gtgtacgacg
     4981 tgcggaagat gatcgccaag agcgagcagg aaatcggcaa ggctaccgcc aagtacttct
     5041 tctacagcaa catcatgaac tttttcaaga ccgagattac cctggccaac ggcgagatcc
     5101 ggaagcggcc tctgatcgag acaaacggcg aaaccgggga gatcgtgtgg gataagggcc
     5161 gggattttgc caccgtgcgg aaagtgctga gcatgcccca agtgaatatc gtgaaaaaga
     5221 ccgaggtgca gacaggcggc ttcagcaaag agtctatcct gcccaagagg aacagcgata
     5281 agctgatcgc cagaaagaag gactgggacc ctaagaagta cggcggcttc gacagcccca
     5341 ccgtggccta ttctgtgctg gtggtggcca aagtggaaaa gggcaagtcc aagaaactga
     5401 agagtgtgaa agagctgctg gggatcacca tcatggaaag aagcagcttc gagaagaatc
     5461 ccatcgactt tctggaagcc aagggctaca aagaagtgaa aaaggacctg atcatcaagc
     5521 tgcctaagta ctccctgttc gagctggaaa acggccggaa gagaatgctg gcctctgccg
     5581 gcgaactgca gaagggaaac gaactggccc tgccctccaa atatgtgaac ttcctgtacc
     5641 tggccagcca ctatgagaag ctgaagggct cccccgagga taatgagcag aaacagctgt
     5701 ttgtggaaca gcacaagcac tacctggacg agatcatcga gcagatcagc gagttctcca
     5761 agagagtgat cctggccgac gctaatctgg acaaagtgct gtccgcctac aacaagcacc
     5821 gggataagcc catcagagag caggccgaga atatcatcca cctgtttacc ctgaccaatc
     5881 tgggagcccc tgccgccttc aagtactttg acaccaccat cgaccggaag aggtacacca
     5941 gcaccaaaga ggtgctggac gccaccctga tccaccagag catcaccggc ctgtacgaga
     6001 cacggatcga cctgtctcag ctgggaggcg acggatccga atccggcagt ggagagggca
     6061 gaggaagtct gctaacatgc ggtgacgtcg aggagaatcc tggcccaatg gtgagcaagg
     6121 gcgaggagga taacatggcc atcatcaagg agttcatgcg cttcaaggtg cacatggagg
     6181 gctccgtgaa cggccacgag ttcgagatcg agggcgaggg cgagggccgc ccctacgagg
     6241 gcacccagac cgccaagctg aaggtgacca agggtggccc cctgcccttc gcctgggaca
     6301 tcctgtcccc tcagttcatg tacggctcca aggcctacgt gaagcacccc gccgacatcc
     6361 ccgactactt gaagctgtcc ttccccgagg gcttcaagtg ggagcgcgtg atgaacttcg
     6421 aggacggcgg cgtggtgacc gtgacccagg actcctccct gcaggacggc gagttcatct
     6481 acaaggtgaa gctgcgcggc accaacttcc cctccgacgg ccccgtaatg cagaagaaaa
     6541 ccatgggctg ggaggcctcc tccgagcgga tgtaccccga ggacggcgcc ctgaagggcg
     6601 agatcaagca gaggctgaag ctgaaggacg gcggccacta cgacgctgag gtcaagacca
     6661 cctacaaggc caagaagccc gtgcagctgc ccggcgccta caacgtcaac atcaagttgg
     6721 acatcacctc ccacaacgag gactacacca tcgtggaaca gtacgaacgc gccgagggcc
     6781 gccactccac cggcggcatg gacgagctgt acaagtaaga attcctagag ctcgctgatc
     6841 agcctcgact gtgccttcta gttgccagcc atctgttgtt tgcccctccc ccgtgccttc
     6901 cttgaccctg gaaggtgcca ctcccactgt cctttcctaa taaaatgagg aaattgcatc
     6961 gcattgtctg agtaggtgtc attctattct ggggggtggg gtggggcagg acagcaaggg
     7021 ggaggattgg gaagagaata gcaggcatgc tggggagcgg ccgcaggaac ccctagtgat
     7081 ggagttggcc actccctctc tgcgcgctcg ctcgctcact gaggccgggc gaccaaaggt
     7141 cgcccgacgc ccgggctttg cccgggcggc ctcagtgagc gagcgagcgc gcagctgcct
     7201 gcaggggcgc ctgatgcggt attttctcct tacgcatctg tgcggtattt cacaccgcat
     7261 acgtcaaagc aaccatagta cgcgccctgt agcggcgcat taagcgcggc gggtgtggtg
     7321 gttacgcgca gcgtgaccgc tacacttgcc agcgccctag cgcccgctcc tttcgctttc
     7381 ttcccttcct ttctcgccac gttcgccggc tttccccgtc aagctctaaa tcgggggctc
     7441 cctttagggt tccgatttag tgctttacgg cacctcgacc ccaaaaaact tgatttgggt
     7501 gatggttcac gtagtgggcc atcgccctga tagacggttt ttcgcccttt gacgttggag
     7561 tccacgttct ttaatagtgg actcttgttc caaactggaa caacactcaa ccctatctcg
     7621 ggctattctt ttgatttata agggattttg ccgatttcgg cctattggtt aaaaaatgag
     7681 ctgatttaac aaaaatttaa cgcgaatttt aacaaaatat taacgtttac aattttatgg
     7741 tgcactctca gtacaatctg ctctgatgcc gcatagttaa gccagccccg acacccgcca
     7801 acacccgctg acgcgccctg acgggcttgt ctgctcccgg catccgctta cagacaagct
     7861 gtgaccgtct ccgggagctg catgtgtcag aggttttcac cgtcatcacc gaaacgcgcg
     7921 agacgaaagg gcctcgtgat acgcctattt ttataggtta atgtcatgat aataatggtt
     7981 tcttagacgt caggtggcac ttttcgggga aatgtgcgcg gaacccctat ttgtttattt
     8041 ttctaaatac attcaaatat gtatccgctc atgagacaat aaccctgata aatgcttcaa
     8101 taatattgaa aaaggaagag tatgagtatt caacatttcc gtgtcgccct tattcccttt
     8161 tttgcggcat tttgccttcc tgtttttgct cacccagaaa cgctggtgaa agtaaaagat
     8221 gctgaagatc agttgggtgc acgagtgggt tacatcgaac tggatctcaa cagcggtaag
     8281 atccttgaga gttttcgccc cgaagaacgt tttccaatga tgagcacttt taaagttctg
     8341 ctatgtggcg cggtattatc ccgtattgac gccgggcaag agcaactcgg tcgccgcata
     8401 cactattctc agaatgactt ggttgagtac tcaccagtca cagaaaagca tcttacggat
     8461 ggcatgacag taagagaatt atgcagtgct gccataacca tgagtgataa cactgcggcc
     8521 aacttacttc tgacaacgat cggaggaccg aaggagctaa ccgctttttt gcacaacatg
     8581 ggggatcatg taactcgcct tgatcgttgg gaaccggagc tgaatgaagc cataccaaac
     8641 gacgagcgtg acaccacgat gcctgtagca atggcaacaa cgttgcgcaa actattaact
     8701 ggcgaactac ttactctagc ttcccggcaa caattaatag actggatgga ggcggataaa
     8761 gttgcaggac cacttctgcg ctcggccctt ccggctggct ggtttattgc tgataaatct
     8821 ggagccggtg agcgtggaag ccgcggtatc attgcagcac tggggccaga tggtaagccc
     8881 tcccgtatcg tagttatcta cacgacgggg agtcaggcaa ctatggatga acgaaataga
     8941 cagatcgctg agataggtgc ctcactgatt aagcattggt aactgtcaga ccaagtttac
     9001 tcatatatac tttagattga tttaaaactt catttttaat ttaaaaggat ctaggtgaag
     9061 atcctttttg ataatctcat gaccaaaatc ccttaacgtg agttttcgtt ccactgagcg
     9121 tcagaccccg tagaaaagat caaaggatct tc
//
