LOCUS       sequence-166590-       10425 bp    DNA     circular SYN 14-APR-2025
DEFINITION  All in one plasmid that expresses Csy4, S.pyogenes FokI-dCas9-NLS, 
            and GFP. Also encodes for multiplexed gRNAs. Backbone derived from 
            pSQT1601 (Addgene #53369)..
ACCESSION   .
VERSION     .
KEYWORDS    .
SOURCE      synthetic DNA construct
  ORGANISM  synthetic DNA construct
REFERENCE   1  (bases 1 to 10425)
  AUTHORS   Havlicek S, Shen Y, Alpagu Y, Bruntraeger MB, Zufir NB, Phuah ZY, Fu
            Z, Dunn NR, Stanton LW
  TITLE     Re-engineered RNA-Guided FokI-Nucleases for Improved Genome Editing 
            in Human Cells.
  JOURNAL   Mol Ther. 2017 Feb 1;25(2):342-355. doi: 
            10.1016/j.ymthe.2016.11.007.
  PUBMED    28153087
REFERENCE   2  (bases 1 to 10425)
  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"; doi: 
            "10.1016/j.ymthe.2016.11"; journalName: "Mol Ther"; date: 
            "2017-02-1- 1"; volume: "25"; issue: "2"; pages: "342-355"
COMMENT     Sequence Label: pSH-Csy4-T2A-SpRFN-P2A-GFP-multi-gRNA
FEATURES             Location/Qualifiers
     source          1..10425
                     /mol_type="other DNA"
                     /organism="synthetic DNA construct"
     promoter        96..344
                     /label=U6 promoter
                     /note="RNA polymerase III promoter for human U6 snRNA 
                     (Domitrovich & Kunkel, 2003)"
     primer_bind     96..116
                     /label=hU6-F
                     /note="Human U6 promoter, forward primer"
     primer_bind     267..286
                     /label=LKO.1 5'
                     /note="Human U6 promoter, forward primer"
     protein_bind    345..364
                     /label=Csy4 Site
                     /bound_moiety="Pseudomonas aeruginosa Csy4 (Cas6f)"
                     /note="cleavage site for the Csy4 endoribonuclease (Cermak 
                     et al. 2017)"
     misc_RNA        386..461
                     /label=gRNA scaffold
                     /note="guide RNA scaffold for the Streptococcus pyogenes 
                     CRISPR/Cas9 system"
     protein_bind    462..481
                     /label=Csy4 Site
                     /bound_moiety="Pseudomonas aeruginosa Csy4 (Cas6f)"
                     /note="cleavage site for the Csy4 endoribonuclease (Cermak 
                     et al. 2017)"
     enhancer        498..801
                     /label=CMV enhancer
                     /note="human cytomegalovirus immediate early enhancer"
     promoter        802..1005
                     /label=CMV promoter
                     /note="human cytomegalovirus (CMV) immediate early 
                     promoter"
     primer_bind     956..976
                     /label=CMV-F
                     /note="Human CMV immediate early promoter, forward primer"
     primer_bind     1002..1026
                     /label=LNCX
                     /note="Human CMV promoter, forward primer"
     regulatory      1061..1070
                     /label=Kozak sequence
                     /note="vertebrate consensus sequence for strong initiation 
                     of translation (Kozak, 1987)"
                     /regulatory_class="other"
     CDS             1631..1684
                     /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             1688..1708
                     /codon_start=1
                     /product="nuclear localization signal of SV40 (simian virus
                     40) large T antigen"
                     /label=SV40 NLS
                     /translation="PKKKRKV"
     CDS             1715..2302
                     /codon_start=1
                     /product="nonspecific DNA cleavage domain of the FokI 
                     endonuclease (Li et al., 1992)"
                     /label=FokI cleavage domain
                     /note="must dimerize to be catalytically active"
                     /translation="QLVKSELEEKKSELRHKLKYVPHEYIELIEIARNSTQDRILEMKV
                     MEFFMKVYGYRGKHLGGSRKPDGAIYTVGSPIDYGVIVDTKAYSGGYNLPIGQADEMQR
                     YVEENQTRNKHINPNEWWKVYPSSVTEFKFLFVSGHFKGNYKAQLTRLNHITNCNGAVL
                     SVEELLIGGEMIKAGTLTLEEVRRKFNNGEINF"
     CDS             join(2378..5776,5777..5779,5780..6376,6377,6378..6380,
                     6381..6382,6383..6385,6386..6478)
                     /codon_start=1
                     /product="Streptococcus pyogenes Cas9 endonuclease with the
                     D1135V/R1335Q/T1337R mutations"
                     /label=Cas9 VQR
                     /note="has an altered recognition specificity for the 
                     protospacer adjacent motif (PAM) (Kleinstiver et al., 
                     2015)"
                     /translation="DKKYSIGLAIGTNSVGWAVITDEYKVPSKKFKVLGNTDRHSIKKN
                     LIGALLFDSGETAEATRLKRTARRRYTRRKNRICYLQEIFSNEMAKVDDSFFHRLEESF
                     LVEEDKKHERHPIFGNIVDEVAYHEKYPTIYHLRKKLVDSTDKADLRLIYLALAHMIKF
                     RGHFLIEGDLNPDNSDVDKLFIQLVQTYNQLFEENPINASGVDAKAILSARLSKSRRLE
                     NLIAQLPGEKKNGLFGNLIALSLGLTPNFKSNFDLAEDAKLQLSKDTYDDDLDNLLAQI
                     GDQYADLFLAAKNLSDAILLSDILRVNTEITKAPLSASMIKRYDEHHQDLTLLKALVRQ
                     QLPEKYKEIFFDQSKNGYAGYIDGGASQEEFYKFIKPILEKMDGTEELLVKLNREDLLR
                     KQRTFDNGSIPHQIHLGELHAILRRQEDFYPFLKDNREKIEKILTFRIPYYVGPLARGN
                     SRFAWMTRKSEETITPWNFEEVVDKGASAQSFIERMTNFDKNLPNEKVLPKHSLLYEYF
                     TVYNELTKVKYVTEGMRKPAFLSGEQKKAIVDLLFKTNRKVTVKQLKEDYFKKIECFDS
                     VEISGVEDRFNASLGTYHDLLKIIKDKDFLDNEENEDILEDIVLTLTLFEDREMIEERL
                     KTYAHLFDDKVMKQLKRRRYTGWGRLSRKLINGIRDKQSGKTILDFLKSDGFANRNFMQ
                     LIHDDSLTFKEDIQKAQVSGQGDSLHEHIANLAGSPAIKKGILQTVKVVDELVKVMGRH
                     KPENIVIEMARENQTTQKGQKNSRERMKRIEEGIKELGSQILKEHPVENTQLQNEKLYL
                     YYLQNGRDMYVDQELDINRLSDYDVDAIVPQSFLKDDSIDNKVLTRSDKNRGKSDNVPS
                     EEVVKKMKNYWRQLLNAKLITQRKFDNLTKAERGGLSELDKAGFIKRQLVETRQITKHV
                     AQILDSRMNTKYDENDKLIREVKVITLKSKLVSDFRKDFQFYKVREINNYHHAHDAYLN
                     AVVGTALIKKYPKLESEFVYGDYKVYDVRKMIAKSEQEIGKATAKYFFYSNIMNFFKTE
                     ITLANGEIRKRPLIETNGETGEIVWDKGRDFATVRKVLSMPQVNIVKKTEVQTGGFSKE
                     SILPKRNSDKLIARKKDWDPKKYGGFDSPTVAYSVLVVAKVEKGKSKKLKSVKELLGIT
                     IMERSSFEKNPIDFLEAKGYKEVKKDLIIKLPKYSLFELENGRKRMLASAGELQKGNEL
                     ALPSKYVNFLYLASHYEKLKGSPEDNEQKQLFVEQHKHYLDEIIEQISEFSKRVILADA
                     NLDKVLSAYNKHRDKPIREQAENIIHLFTLTNLGAPAAFKYFDTTIDRKRYTSTKEVLD
                     ATLIHQSITGLYETRIDLSQLGGD"
     CDS             join(2378..2401,2402..2404,2405..4891,4892..4894,
                     4895..6478)
                     /codon_start=1
                     /product="catalytically dead mutant of the Cas9 
                     endonuclease from the Streptococcus pyogenes Type II 
                     CRISPR/Cas system"
                     /label=dCas9
                     /note="RNA-guided DNA-binding protein that lacks 
                     endonuclease activity due to the D10A mutation in the RuvC 
                     catalytic domain and the H840A mutation in the HNH 
                     catalytic domain"
                     /translation="DKKYSIGLAIGTNSVGWAVITDEYKVPSKKFKVLGNTDRHSIKKN
                     LIGALLFDSGETAEATRLKRTARRRYTRRKNRICYLQEIFSNEMAKVDDSFFHRLEESF
                     LVEEDKKHERHPIFGNIVDEVAYHEKYPTIYHLRKKLVDSTDKADLRLIYLALAHMIKF
                     RGHFLIEGDLNPDNSDVDKLFIQLVQTYNQLFEENPINASGVDAKAILSARLSKSRRLE
                     NLIAQLPGEKKNGLFGNLIALSLGLTPNFKSNFDLAEDAKLQLSKDTYDDDLDNLLAQI
                     GDQYADLFLAAKNLSDAILLSDILRVNTEITKAPLSASMIKRYDEHHQDLTLLKALVRQ
                     QLPEKYKEIFFDQSKNGYAGYIDGGASQEEFYKFIKPILEKMDGTEELLVKLNREDLLR
                     KQRTFDNGSIPHQIHLGELHAILRRQEDFYPFLKDNREKIEKILTFRIPYYVGPLARGN
                     SRFAWMTRKSEETITPWNFEEVVDKGASAQSFIERMTNFDKNLPNEKVLPKHSLLYEYF
                     TVYNELTKVKYVTEGMRKPAFLSGEQKKAIVDLLFKTNRKVTVKQLKEDYFKKIECFDS
                     VEISGVEDRFNASLGTYHDLLKIIKDKDFLDNEENEDILEDIVLTLTLFEDREMIEERL
                     KTYAHLFDDKVMKQLKRRRYTGWGRLSRKLINGIRDKQSGKTILDFLKSDGFANRNFMQ
                     LIHDDSLTFKEDIQKAQVSGQGDSLHEHIANLAGSPAIKKGILQTVKVVDELVKVMGRH
                     KPENIVIEMARENQTTQKGQKNSRERMKRIEEGIKELGSQILKEHPVENTQLQNEKLYL
                     YYLQNGRDMYVDQELDINRLSDYDVDAIVPQSFLKDDSIDNKVLTRSDKNRGKSDNVPS
                     EEVVKKMKNYWRQLLNAKLITQRKFDNLTKAERGGLSELDKAGFIKRQLVETRQITKHV
                     AQILDSRMNTKYDENDKLIREVKVITLKSKLVSDFRKDFQFYKVREINNYHHAHDAYLN
                     AVVGTALIKKYPKLESEFVYGDYKVYDVRKMIAKSEQEIGKATAKYFFYSNIMNFFKTE
                     ITLANGEIRKRPLIETNGETGEIVWDKGRDFATVRKVLSMPQVNIVKKTEVQTGGFSKE
                     SILPKRNSDKLIARKKDWDPKKYGGFDSPTVAYSVLVVAKVEKGKSKKLKSVKELLGIT
                     IMERSSFEKNPIDFLEAKGYKEVKKDLIIKLPKYSLFELENGRKRMLASAGELQKGNEL
                     ALPSKYVNFLYLASHYEKLKGSPEDNEQKQLFVEQHKHYLDEIIEQISEFSKRVILADA
                     NLDKVLSAYNKHRDKPIREQAENIIHLFTLTNLGAPAAFKYFDTTIDRKRYTSTKEVLD
                     ATLIHQSITGLYETRIDLSQLGGD"
     CDS             6485..6505
                     /codon_start=1
                     /product="nuclear localization signal of SV40 (simian virus
                     40) large T antigen"
                     /label=SV40 NLS
                     /translation="PKKKRKV"
     CDS             6512..6577
                     /codon_start=1
                     /product="three tandem FLAG(R) epitope tags, followed by an
                     enterokinase cleavage site"
                     /label=3xFLAG
                     /translation="DYKDHDGDYKDHDIDYKDDDDK"
     CDS             6587..6643
                     /codon_start=1
                     /product="2A peptide from porcine teschovirus-1 
                     polyprotein"
                     /label=P2A
                     /note="Eukaryotic ribosomes fail to insert a peptide bond 
                     between the Gly and Pro residues, yielding separate 
                     polypeptides."
                     /translation="ATNFSLLKQAGDVEENPGP"
     CDS             join(6644..6646,6647..7360)
                     /codon_start=1
                     /product="the original enhanced GFP (Yang et al., 1996)"
                     /label=EGFP
                     /note="mammalian codon-optimized"
                     /translation="VSKGEELFTGVVPILVELDGDVNGHKFSVSGEGEGDATYGKLTLK
                     FICTTGKLPVPWPTLVTTLTYGVQCFSRYPDHMKQHDFFKSAMPEGYVQERTIFFKDDG
                     NYKTRAEVKFEGDTLVNRIELKGIDFKEDGNILGHKLEYNYNSHNVYIMADKQKNGIKV
                     NFKIRHNIEDGSVQLADHYQQNTPIGDGPVLLPDNHYLSTQSALSKDPNEKRDHMVLLE
                     FVTAAGITLGMDELYK"
     CDS             join(6644..6646,6647..7357)
                     /codon_start=1
                     /product="enhanced GFP"
                     /label=EGFP
                     /translation="VSKGEELFTGVVPILVELDGDVNGHKFSVSGEGEGDATYGKLTLK
                     FICTTGKLPVPWPTLVTTLTYGVQCFSRYPDHMKQHDFFKSAMPEGYVQERTIFFKDDG
                     NYKTRAEVKFEGDTLVNRIELKGIDFKEDGNILGHKLEYNYNSHNVYIMADKQKNGIKV
                     NFKIRHNIEDGSVQLADHYQQNTPIGDGPVLLPDNHYLSTQSALSKDPNEKRDHMVLLE
                     FVTAAGITLGMDELYK"
     primer_bind     complement(6686..6707)
                     /label=EGFP-N
                     /note="EGFP, reverse primer"
     primer_bind     complement(6947..6966)
                     /label=EXFP-R
                     /note="For distinguishing EGFP variants, reverse primer"
     primer_bind     7294..7315
                     /label=EGFP-C
                     /note="EGFP, forward primer"
     primer_bind     complement(7447..7466)
                     /label=Bglob-pA-R
                     /note="Rabbit beta-globin polyA region, reverse primer"
     polyA_signal    7512..7567
                     /label=beta-globin poly(A) signal
                     /note="rabbit beta-globin polyadenylation signal (Gil and 
                     Proudfoot, 1987)"
     primer_bind     complement(7566..7585)
                     /label=rbglobpA-R
                     /note="Rabbit beta-globin polyA, reverse primer. Also 
                     called rb-glob-pA-term-R"
     primer_bind     complement(7928..7944)
                     /label=M13 rev
                     /note="common sequencing primer, one of multiple similar 
                     variants"
     primer_bind     complement(7928..7944)
                     /label=M13 Reverse
                     /note="In lacZ gene. Also called M13-rev"
     primer_bind     complement(7941..7963)
                     /label=M13/pUC Reverse
                     /note="In lacZ gene"
     protein_bind    7952..7968
                     /label=lac operator
                     /bound_moiety="lac repressor encoded by lacI"
                     /note="The lac repressor binds to the lac operator to 
                     inhibit transcription in E. coli. This inhibition can be 
                     relieved by adding lactose or 
                     isopropyl-beta-D-thiogalactopyranoside (IPTG)."
     promoter        complement(join(7976..7982,7983..8000,8001..8006))
                     /label=lac promoter
                     /note="promoter for the E. coli lac operon"
     protein_bind    8021..8042
                     /label=CAP binding site
                     /bound_moiety="E. coli catabolite activator protein"
                     /note="CAP binding activates transcription in the presence 
                     of cAMP."
     promoter        8101..8296
                     /label=SV40 promoter
                     /note="SV40 early promoter"
     rep_origin      8147..8282
                     /label=SV40 ori
                     /note="SV40 origin of replication"
     polyA_signal    8302..8436
                     /label=SV40 poly(A) signal
                     /note="SV40 polyadenylation signal"
     primer_bind     complement(8339..8358)
                     /label=SV40pA-R
                     /note="SV40 polyA, reverse primer"
     primer_bind     8393..8412
                     /label=EBV-rev
                     /note="SV40 polyA terminator, reverse primer"
     primer_bind     complement(8504..8521)
                     /label=L4440
                     /note="L4440 vector, forward primer"
     rep_origin      complement(8675..9263)
                     /direction=LEFT
                     /label=ori
                     /note="high-copy-number ColE1/pMB1/pBR322/pUC origin of 
                     replication"
     primer_bind     complement(8755..8774)
                     /label=pBR322ori-F
                     /note="pBR322 origin, forward primer"
     CDS             complement(join(9434..10225,10226..10294))
                     /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     10057..10076
                     /label=Amp-R
                     /note="Ampicillin resistance gene, reverse primer"
     promoter        complement(10295..10399)
                     /gene="bla"
                     /label=AmpR promoter
ORIGIN
        1 gtcgacattg attattgatc gctagctgta caaaaaagca ggctttaaag gaaccaattc
       61 agtcgactgg atccggtacc aaggtcgggc aggaagaggg cctatttccc atgattcctt
      121 catatttgca tatacgatac aaggctgtta gagagataat tagaattaat ttgactgtaa
      181 acacaaagat attagtacaa aatacgtgac gtagaaagta ataatttctt gggtagtttg
      241 cagttttaaa attatgtttt aaaatggact atcatatgct taccgtaact tgaaagtatt
      301 tcgatttctt ggctttatat atcttgtgga aaggacgaaa caccgttcac tgccgtatag
      361 gcagtgagac gattaatgcg tctcagtttt agagctagaa atagcaagtt aaaataaggc
      421 tagtccgtta tcaacttgaa aaagtggcac cgagtcggtg cgttcactgc cgtataggca
      481 gtttttttaa gcttccgcgt tacataactt acggtaaatg gcccgcctgg ctgaccgccc
      541 aacgaccccc gcccattgac gtcaataatg acgtatgttc ccatagtaac gccaataggg
      601 actttccatt gacgtcaatg ggtggagtat ttacggtaaa ctgcccactt ggcagtacat
      661 caagtgtatc atatgccaag tacgccccct attgacgtca atgacggtaa atggcccgcc
      721 tggcattatg cccagtacat gaccttatgg gactttccta cttggcagta catctacgta
      781 ttagtcatcg ctattaccat ggtgatgcgg ttttggcagt acatcaatgg gcgtggatag
      841 cggtttgact cacggggatt tccaagtctc caccccattg acgtcaatgg gagtttgttt
      901 tggcaccaaa atcaacggga ctttccaaaa tgtcgtaaca actccgcccc attgacgcaa
      961 atgggcggta ggcgtgtacg gtgggaggtc tatataagca gagctcgttt agtgaaccgt
     1021 cagatcgcct ggagtaccgc gggcccggga tccaccggtc gccaccatgg gtgatcatta
     1081 tctggatatt cggctgaggc ctgatccaga gttcccacct gcgcagctga tgtctgtcct
     1141 ttttggcaaa cttcatcagg ccctggttgc ccagggcgga gatcggatag gggtaagctt
     1201 tccagacctc gacgaaagcc ggagccgcct gggagaacgc ctgcggatcc acgcttctgc
     1261 cgacgatctg agagccttgc tggcaaggcc atggcttgag gggctccggg atcacctgca
     1321 gtttggcgaa cccgccgttg ttccccaccc aaccccttat cggcaggtgt ctagagtgca
     1381 ggccaaatct aatccagaac ggctgcgacg gcgactcatg cggcgacatg atcttagcga
     1441 ggaagaggcc cgaaaaagaa tccctgatac cgtggcccgc gcccttgact tgccttttgt
     1501 cacactgcgg tcccagagta cggggcagca tttcagactt ttcattcgac acgggccact
     1561 gcaagttacc gccgaagaag gaggctttac ttgttatgga ctctccaagg gaggtttcgt
     1621 gccctggttt gagggcagag gaagtctgtt aacatgcggt gacgtcgagg agaatcctgg
     1681 cccaatgcct aagaagaagc ggaaggtgag cagccaactt gtgaagtctg aactcgagga
     1741 gaaaaaatca gagttgagac acaagttgaa gtacgtgcca cacgaataca tcgagcttat
     1801 cgagatcgcc agaaacagta cccaggatag gatccttgag atgaaagtca tggagttctt
     1861 tatgaaggtc tacggttata gaggaaagca ccttggcggt agcagaaagc ccgatggcgc
     1921 catctatact gtcggatctc ctatcgatta tggggtgatc gtggatacca aagcttactc
     1981 aggcgggtac aacttgccca taggacaagc cgacgagatg cagcggtatg tcgaagagaa
     2041 ccagacgcgc aacaagcaca tcaaccccaa tgaatggtgg aaagtgtacc caagtagtgt
     2101 gactgagttc aagttcctgt ttgtctccgg ccactttaag ggcaattata aagctcagct
     2161 cactagactc aatcacatca caaactgcaa cggagctgtg ttgtcagtgg aggagctcct
     2221 gattggaggc gagatgatca aagccggcac ccttacactg gaggaggtgc ggcggaagtt
     2281 caacaatgga gagatcaact tcggtggcgg tggatccggt ggcggtggat ccggtggcgg
     2341 tggatccggt ggcggtggat ccggtggtgg tggatccgat aaaaagtatt ctattggttt
     2401 agccatcggc actaattccg ttggatgggc tgtcataacc gatgaataca aagtaccttc
     2461 aaagaaattt aaggtgttgg ggaacacaga ccgtcattcg attaaaaaga atcttatcgg
     2521 tgccctccta ttcgatagtg gcgaaacggc agaggcgact cgcctgaaac gaaccgctcg
     2581 gagaaggtat acacgtcgca agaaccgaat atgttactta caagaaattt ttagcaatga
     2641 gatggccaaa gttgacgatt ctttctttca ccgtttggaa gagtccttcc ttgtcgaaga
     2701 ggacaagaaa catgaacggc accccatctt tggaaacata gtagatgagg tggcatatca
     2761 tgaaaagtac ccaacgattt atcacctcag aaaaaagcta gttgactcaa ctgataaagc
     2821 ggacctgagg ttaatctact tggctcttgc ccatatgata aagttccgtg ggcactttct
     2881 cattgagggt gatctaaatc cggacaactc ggatgtcgac aaactgttca tccagttagt
     2941 acaaacctat aatcagttgt ttgaagagaa ccctataaat gcaagtggcg tggatgcgaa
     3001 ggctattctt agcgcccgcc tctctaaatc ccgacggcta gaaaacctga tcgcacaatt
     3061 acccggagag aagaaaaatg ggttgttcgg taaccttata gcgctctcac taggcctgac
     3121 accaaatttt aagtcgaact tcgacttagc tgaagatgcc aaattgcagc ttagtaagga
     3181 cacgtacgat gacgatctcg acaatctact ggcacaaatt ggagatcagt atgcggactt
     3241 atttttggct gccaaaaacc ttagcgatgc aatcctccta tctgacatac tgagagttaa
     3301 tactgagatt accaaggcgc cgttatccgc ttcaatgatc aaaaggtacg atgaacatca
     3361 ccaagacttg acacttctca aggccctagt ccgtcagcaa ctgcctgaga aatataagga
     3421 aatattcttt gatcagtcga aaaacgggta cgcaggttat attgacggcg gagcgagtca
     3481 agaggaattc tacaagttta tcaaacccat attagagaag atggatggga cggaagagtt
     3541 gcttgtaaaa ctcaatcgcg aagatctact gcgaaagcag cggactttcg acaacggtag
     3601 cattccacat caaatccact taggcgaatt gcatgctata cttagaaggc aggaggattt
     3661 ttatccgttc ctcaaagaca atcgtgaaaa gattgagaaa atcctaacct ttcgcatacc
     3721 ttactatgtg ggacccctgg cccgagggaa ctctcggttc gcatggatga caagaaagtc
     3781 cgaagaaacg attactccat ggaattttga ggaagttgtc gataaaggtg cgtcagctca
     3841 atcgttcatc gagaggatga ccaactttga caagaattta ccgaacgaaa aagtattgcc
     3901 taagcacagt ttactttacg agtatttcac agtgtacaat gaactcacga aagttaagta
     3961 tgtcactgag ggcatgcgta aacccgcctt tctaagcgga gaacagaaga aagcaatagt
     4021 agatctgtta ttcaagacca accgcaaagt gacagttaag caattgaaag aggactactt
     4081 taagaaaatt gaatgcttcg attctgtcga gatctccggg gtagaagatc gatttaatgc
     4141 gtcacttggt acgtatcatg acctcctaaa gataattaaa gataaggact tcctggataa
     4201 cgaagagaat gaagatatct tagaagatat agtgttgact cttaccctct ttgaagatcg
     4261 ggaaatgatt gaggaaagac taaaaacata cgctcacctg ttcgacgata aggttatgaa
     4321 acagttaaag aggcgtcgct atacgggctg gggacgattg tcgcggaaac ttatcaacgg
     4381 gataagagac aagcaaagtg gtaaaactat tctcgatttt ctaaagagcg acggcttcgc
     4441 caataggaac tttatgcagc tgatccatga tgactcttta accttcaaag aggatataca
     4501 aaaggcacag gtttccggac aaggggactc attgcacgaa catattgcga atcttgctgg
     4561 ttcgccagcc atcaaaaagg gcatactcca gacagtcaaa gtagtggatg agctagttaa
     4621 ggtcatggga cgtcacaaac cggaaaacat tgtaatcgag atggcacgcg aaaatcaaac
     4681 gactcagaag gggcaaaaaa acagtcgaga gcggatgaag agaatagaag agggtattaa
     4741 agaactgggc agccagatct taaaggagca tcctgtggaa aatacccaat tgcagaacga
     4801 gaaactttac ctctattacc tacaaaatgg aagggacatg tatgttgatc aggaactgga
     4861 cataaaccgt ttatctgatt acgacgtcga tgccattgta ccccaatcct ttttgaagga
     4921 cgattcaatc gacaataaag tgcttacacg ctcggataag aaccgaggga aaagtgacaa
     4981 tgttccaagc gaggaagtcg taaagaaaat gaagaactat tggcggcagc tcctaaatgc
     5041 gaaactgata acgcaaagaa agttcgataa cttaactaaa gctgagaggg gtggcttgtc
     5101 tgaacttgac aaggccggat ttattaaacg tcagctcgtg gaaacccgcc aaatcacaaa
     5161 gcatgttgca cagatactag attcccgaat gaatacgaaa tacgacgaga acgataagct
     5221 gattcgggaa gtcaaagtaa tcactttaaa gtcaaaattg gtgtcggact tcagaaagga
     5281 ttttcaattc tataaagtta gggagataaa taactaccac catgcgcacg acgcttatct
     5341 taatgccgtc gtagggaccg cactcattaa gaaatacccg aagctagaaa gtgagtttgt
     5401 gtatggtgat tacaaagttt atgacgtccg taagatgatc gcgaaaagcg aacaggagat
     5461 aggcaaggct acagccaaat acttctttta ttctaacatt atgaatttct ttaagacgga
     5521 aatcactctg gcaaacggag agatacgcaa acgaccttta attgaaacca atggggagac
     5581 aggtgaaatc gtatgggata agggccggga cttcgcgacg gtgagaaaag ttttgtccat
     5641 gccccaagtc aacatagtaa agaaaactga ggtgcagacc ggagggtttt caaaggaatc
     5701 gattcttcca aaaaggaata gtgataagct catcgctcgt aaaaaggact gggacccgaa
     5761 aaagtacggt ggcttcgata gccctacagt tgcctattct gtcctagtag tggcaaaagt
     5821 tgagaaggga aaatccaaga aactgaagtc agtcaaagaa ttattgggga taacgattat
     5881 ggagcgctcg tcttttgaaa agaaccccat cgacttcctt gaggcgaaag gttacaagga
     5941 agtaaaaaag gatctcataa ttaaactacc aaagtatagt ctgtttgagt tagaaaatgg
     6001 ccgaaaacgg atgttggcta gcgccggaga gcttcaaaag gggaacgaac tcgcactacc
     6061 gtctaaatac gtgaatttcc tgtatttagc gtcccattac gagaagttga aaggttcacc
     6121 tgaagataac gaacagaagc aactttttgt tgagcagcac aaacattatc tcgacgaaat
     6181 catagagcaa atttcggaat tcagtaagag agtcatccta gctgatgcca atctggacaa
     6241 agtattaagc gcatacaaca agcacaggga taaacccata cgtgagcagg cggaaaatat
     6301 tatccatttg tttactctta ccaacctcgg cgctccagcc gcattcaagt attttgacac
     6361 aacgatagat cgcaaacgat acacttctac caaggaggtg ctagacgcga cactgattca
     6421 ccaatccatc acgggattat atgaaactcg gatagatttg tcacagcttg ggggtgacgg
     6481 atcccccaag aagaagagga aagtctcgag cgactacaaa gaccatgacg gtgattataa
     6541 agatcatgac atcgattaca aggatgacga tgacaaggga agcggagcca ctaacttctc
     6601 cctgttgaaa caagcagggg atgtcgaaga gaatcccggg ccagtgagca agggcgagga
     6661 gctgttcacc ggggtggtgc ccatcctggt cgagctggac ggcgacgtaa acggccacaa
     6721 gttcagcgtg tccggcgagg gcgagggcga tgccacctac ggcaagctga ccctgaagtt
     6781 catctgcacc accggcaagc tgcccgtgcc ctggcccacc ctcgtgacca ccctgaccta
     6841 cggcgtgcag tgcttcagcc gctaccccga ccacatgaag cagcacgact tcttcaagtc
     6901 cgccatgccc gaaggctacg tccaggagcg caccatcttc ttcaaggacg acggcaacta
     6961 caagacccgc gccgaggtga agttcgaggg cgacaccctg gtgaaccgca tcgagctgaa
     7021 gggcatcgac ttcaaggagg acggcaacat cctggggcac aagctggagt acaactacaa
     7081 cagccacaac gtctatatca tggccgacaa gcagaagaac ggcatcaagg tgaacttcaa
     7141 gatccgccac aacatcgagg acggcagcgt gcagctcgcc gaccactacc agcagaacac
     7201 ccccatcggc gacggccccg tgctgctgcc cgacaaccac tacctgagca cccagtccgc
     7261 cctgagcaaa gaccccaacg agaagcgcga tcacatggtc ctgctggagt tcgtgaccgc
     7321 cgccgggatc actctcggca tggacgagct gtacaagtaa agcggccgca ctcctcaggt
     7381 gcaggctgcc tatcagaagg tggtggctgg tgtggccaat gccctggctc acaaatacca
     7441 ctgagatctt tttccctctg ccaaaaatta tggggacatc atgaagcccc ttgagcatct
     7501 gacttctggc taataaagga aatttatttt cattgcaata gtgtgttgga attttttgtg
     7561 tctctcactc ggaaggacat atgggagggc aaatcattta aaacatcaga atgagtattt
     7621 ggtttagagt ttggcaacat atgcccatat gctggctgcc atgaacaaag gttggctata
     7681 aagaggtcat cagtatatga aacagccccc tgctgtccat tccttattcc atagaaaagc
     7741 cttgacttga ggttagattt tttttatatt ttgttttgtg ttattttttt ctttaacatc
     7801 cctaaaattt tccttacatg ttttactagc cagatttttc ctcctctcct gactactccc
     7861 agtcatagct gtccctcttc tcttatggag atccctcgac ctgcagccca agcttggcgt
     7921 aatcatggtc atagctgttt cctgtgtgaa attgttatcc gctcacaatt ccacacaaca
     7981 tacgagccgg aagcataaag tgtaaagcct ggggtgccta atgagtgagc taactcacat
     8041 taattgcgtt gcgctcactg cccgctttcc agtcgggaaa cctgtcgtgc cagcggatcc
     8101 gcatctcaat tagtcagcaa ccatagtccc gcccctaact ccgcccatcc cgcccctaac
     8161 tccgcccagt tccgcccatt ctccgcccca tggctgacta atttttttta tttatgcaga
     8221 ggccgaggcc gcctcggcct ctgagctatt ccagaagtag tgaggaggct tttttggagg
     8281 cctaggcttt tgcaaaaagc taacttgttt attgcagctt ataatggtta caaataaagc
     8341 aatagcatca caaatttcac aaataaagca tttttttcac tgcattctag ttgtggtttg
     8401 tccaaactca tcaatgtatc ttatcatgtc tggatccgct gcattaatga atcggccaac
     8461 gcgcggggag aggcggtttg cgtattgggc gctcttccgc ttcctcgctc actgactcgc
     8521 tgcgctcggt cgttcggctg cggcgagcgg tatcagctca ctcaaaggcg gtaatacggt
     8581 tatccacaga atcaggggat aacgcaggaa agaacatgtg agcaaaaggc cagcaaaagg
     8641 ccaggaaccg taaaaaggcc gcgttgctgg cgtttttcca taggctccgc ccccctgacg
     8701 agcatcacaa aaatcgacgc tcaagtcaga ggtggcgaaa cccgacagga ctataaagat
     8761 accaggcgtt tccccctgga agctccctcg tgcgctctcc tgttccgacc ctgccgctta
     8821 ccggatacct gtccgccttt ctcccttcgg gaagcgtggc gctttctcaa tgctcacgct
     8881 gtaggtatct cagttcggtg taggtcgttc gctccaagct gggctgtgtg cacgaacccc
     8941 ccgttcagcc cgaccgctgc gccttatccg gtaactatcg tcttgagtcc aacccggtaa
     9001 gacacgactt atcgccactg gcagcagcca ctggtaacag gattagcaga gcgaggtatg
     9061 taggcggtgc tacagagttc ttgaagtggt ggcctaacta cggctacact agaaggacag
     9121 tatttggtat ctgcgctctg ctgaagccag ttaccttcgg aaaaagagtt ggtagctctt
     9181 gatccggcaa acaaaccacc gctggtagcg gtggtttttt tgtttgcaag cagcagatta
     9241 cgcgcagaaa aaaaggatct caagaagatc ctttgatctt ttctacgggg tctgacgctc
     9301 agtggaacga aaactcacgt taagggattt tggtcatgag attatcaaaa aggatcttca
     9361 cctagatcct tttaaattaa aaatgaagtt ttaaatcaat ctaaagtata tatgagtaaa
     9421 cttggtctga cagttaccaa tgcttaatca gtgaggcacc tatctcagcg atctgtctat
     9481 ttcgttcatc catagttgcc tgactccccg tcgtgtagat aactacgata cgggagggct
     9541 taccatctgg ccccagtgct gcaatgatac cgcgagaccc acgctcaccg gctccagatt
     9601 tatcagcaat aaaccagcca gccggaaggg ccgagcgcag aagtggtcct gcaactttat
     9661 ccgcctccat ccagtctatt aattgttgcc gggaagctag agtaagtagt tcgccagtta
     9721 atagtttgcg caacgttgtt gccattgcta caggcatcgt ggtgtcacgc tcgtcgtttg
     9781 gtatggcttc attcagctcc ggttcccaac gatcaaggcg agttacatga tcccccatgt
     9841 tgtgcaaaaa agcggttagc tccttcggtc ctccgatcgt tgtcagaagt aagttggccg
     9901 cagtgttatc actcatggtt atggcagcac tgcataattc tcttactgtc atgccatccg
     9961 taagatgctt ttctgtgact ggtgagtact caaccaagtc attctgagaa tagtgtatgc
    10021 ggcgaccgag ttgctcttgc ccggcgtcaa tacgggataa taccgcgcca catagcagaa
    10081 ctttaaaagt gctcatcatt ggaaaacgtt cttcggggcg aaaactctca aggatcttac
    10141 cgctgttgag atccagttcg atgtaaccca ctcgtgcacc caactgatct tcagcatctt
    10201 ttactttcac cagcgtttct gggtgagcaa aaacaggaag gcaaaatgcc gcaaaaaagg
    10261 gaataagggc gacacggaaa tgttgaatac tcatactctt cctttttcaa tattattgaa
    10321 gcatttatca gggttattgt ctcatgagcg gatacatatt tgaatgtatt tagaaaaata
    10381 aacaaatagg ggttccgcgc acatttcccc gaaaagtgcc acctg
//
