LOCUS       sequence-170275-       10426 bp    DNA     circular SYN 15-DEC-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 10426)
  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 10426)
  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: 
            "10.1016/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..10426
                     /mol_type="other DNA"
                     /organism="synthetic DNA construct"
     primer_bind     complement(83..99)
                     /label=M13 rev
                     /note="common sequencing primer, one of multiple similar 
                     variants"
     primer_bind     complement(83..99)
                     /label=M13 Reverse
                     /note="In lacZ gene. Also called M13-rev"
     primer_bind     complement(96..118)
                     /label=M13/pUC Reverse
                     /note="In lacZ gene"
     protein_bind    107..123
                     /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(131..137,138..155,156..161))
                     /label=lac promoter
                     /note="promoter for the E. coli lac operon"
     protein_bind    176..197
                     /label=CAP binding site
                     /bound_moiety="E. coli catabolite activator protein"
                     /note="CAP binding activates transcription in the presence 
                     of cAMP."
     promoter        256..451
                     /label=SV40 promoter
                     /note="SV40 early promoter"
     rep_origin      302..437
                     /label=SV40 ori
                     /note="SV40 origin of replication"
     polyA_signal    457..591
                     /label=SV40 poly(A) signal
                     /note="SV40 polyadenylation signal"
     primer_bind     complement(494..513)
                     /label=SV40pA-R
                     /note="SV40 polyA, reverse primer"
     primer_bind     548..567
                     /label=EBV-rev
                     /note="SV40 polyA terminator, reverse primer"
     primer_bind     complement(659..676)
                     /label=L4440
                     /note="L4440 vector, forward primer"
     rep_origin      complement(830..1418)
                     /direction=LEFT
                     /label=ori
                     /note="high-copy-number ColE1/pMB1/pBR322/pUC origin of 
                     replication"
     primer_bind     complement(910..929)
                     /label=pBR322ori-F
                     /note="pBR322 origin, forward primer"
     CDS             complement(join(1589..2380,2381..2449))
                     /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     2212..2231
                     /label=Amp-R
                     /note="Ampicillin resistance gene, reverse primer"
     promoter        complement(2450..2554)
                     /gene="bla"
                     /label=AmpR promoter
     promoter        2677..2925
                     /label=U6 promoter
                     /note="RNA polymerase III promoter for human U6 snRNA 
                     (Domitrovich & Kunkel, 2003)"
     primer_bind     2677..2697
                     /label=hU6-F
                     /note="Human U6 promoter, forward primer"
     primer_bind     2848..2867
                     /label=LKO.1 5'
                     /note="Human U6 promoter, forward primer"
     protein_bind    2926..2945
                     /label=Csy4 Site
                     /bound_moiety="Pseudomonas aeruginosa Csy4 (Cas6f)"
                     /note="cleavage site for the Csy4 endoribonuclease (Cermak 
                     et al. 2017)"
     misc_RNA        2967..3042
                     /label=gRNA scaffold
                     /note="guide RNA scaffold for the Streptococcus pyogenes 
                     CRISPR/Cas9 system"
     protein_bind    3043..3062
                     /label=Csy4 Site
                     /bound_moiety="Pseudomonas aeruginosa Csy4 (Cas6f)"
                     /note="cleavage site for the Csy4 endoribonuclease (Cermak 
                     et al. 2017)"
     enhancer        3079..3382
                     /label=CMV enhancer
                     /note="human cytomegalovirus immediate early enhancer"
     promoter        3383..3586
                     /label=CMV promoter
                     /note="human cytomegalovirus (CMV) immediate early 
                     promoter"
     primer_bind     3537..3557
                     /label=CMV-F
                     /note="Human CMV immediate early promoter, forward primer"
     primer_bind     3583..3607
                     /label=LNCX
                     /note="Human CMV promoter, forward primer"
     regulatory      3642..3651
                     /label=Kozak sequence
                     /note="vertebrate consensus sequence for strong initiation 
                     of translation (Kozak, 1987)"
                     /regulatory_class="other"
     CDS             4212..4265
                     /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             4269..4289
                     /codon_start=1
                     /product="nuclear localization signal of SV40 (simian virus
                     40) large T antigen"
                     /label=SV40 NLS
                     /translation="PKKKRKV"
     CDS             4296..4883
                     /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(4959..8357,8358..8360,8361..8957,8958,8959..8961,
                     8962..8963,8964..8966,8967..9059)
                     /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(4959..4982,4983..4985,4986..7472,7473..7475,
                     7476..9059)
                     /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             9066..9086
                     /codon_start=1
                     /product="nuclear localization signal of SV40 (simian virus
                     40) large T antigen"
                     /label=SV40 NLS
                     /translation="PKKKRKV"
     CDS             9093..9158
                     /codon_start=1
                     /product="three tandem FLAG(R) epitope tags, followed by an
                     enterokinase cleavage site"
                     /label=3xFLAG
                     /translation="DYKDHDGDYKDHDIDYKDDDDK"
     CDS             9168..9224
                     /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(9225..9227,9228..9941)
                     /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(9225..9227,9228..9938)
                     /codon_start=1
                     /product="enhanced GFP"
                     /label=EGFP
                     /translation="VSKGEELFTGVVPILVELDGDVNGHKFSVSGEGEGDATYGKLTLK
                     FICTTGKLPVPWPTLVTTLTYGVQCFSRYPDHMKQHDFFKSAMPEGYVQERTIFFKDDG
                     NYKTRAEVKFEGDTLVNRIELKGIDFKEDGNILGHKLEYNYNSHNVYIMADKQKNGIKV
                     NFKIRHNIEDGSVQLADHYQQNTPIGDGPVLLPDNHYLSTQSALSKDPNEKRDHMVLLE
                     FVTAAGITLGMDELYK"
     primer_bind     complement(9267..9288)
                     /label=EGFP-N
                     /note="EGFP, reverse primer"
     primer_bind     complement(9528..9547)
                     /label=EXFP-R
                     /note="For distinguishing EGFP variants, reverse primer"
     primer_bind     9875..9896
                     /label=EGFP-C
                     /note="EGFP, forward primer"
     primer_bind     complement(10028..10047)
                     /label=Bglob-pA-R
                     /note="Rabbit beta-globin polyA region, reverse primer"
     polyA_signal    10093..10148
                     /label=beta-globin poly(A) signal
                     /note="rabbit beta-globin polyadenylation signal (Gil and 
                     Proudfoot, 1987)"
     primer_bind     complement(10147..10166)
                     /label=rbglobpA-R
                     /note="Rabbit beta-globin polyA, reverse primer. Also 
                     called rb-glob-pA-term-R"
ORIGIN
        1 ctcctgacta ctcccagtca tagctgtccc tcttctctta tggagatccc tcgacctgca
       61 gcccaagctt ggcgtaatca tggtcatagc tgtttcctgt gtgaaattgt tatccgctca
      121 caattccaca caacatacga gccggaagca taaagtgtaa agcctggggt gcctaatgag
      181 tgagctaact cacattaatt gcgttgcgct cactgcccgc tttccagtcg ggaaacctgt
      241 cgtgccagcg gatccgcatc tcaattagtc agcaaccata gtcccgcccc taactccgcc
      301 catcccgccc ctaactccgc ccagttccgc ccattctccg ccccatggct gactaatttt
      361 ttttatttat gcagaggccg aggccgcctc ggcctctgag ctattccaga agtagtgagg
      421 aggctttttt ggaggcctag gcttttgcaa aaagctaact tgtttattgc agcttataat
      481 ggttacaaat aaagcaatag catcacaaat ttcacaaata aagcattttt ttcactgcat
      541 tctagttgtg gtttgtccaa actcatcaat gtatcttatc atgtctggat ccgctgcatt
      601 aatgaatcgg ccaacgcgcg gggagaggcg gtttgcgtat tgggcgctct tccgcttcct
      661 cgctcactga ctcgctgcgc tcggtcgttc ggctgcggcg agcggtatca gctcactcaa
      721 aggcggtaat acggttatcc acagaatcag gggataacgc aggaaagaac atgtgagcaa
      781 aaggccagca aaaggccagg aaccgtaaaa aggccgcgtt gctggcgttt ttccataggc
      841 tccgcccccc tgacgagcat cacaaaaatc gacgctcaag tcagaggtgg cgaaacccga
      901 caggactata aagataccag gcgtttcccc ctggaagctc cctcgtgcgc tctcctgttc
      961 cgaccctgcc gcttaccgga tacctgtccg cctttctccc ttcgggaagc gtggcgcttt
     1021 ctcatagctc acgctgtagg tatctcagtt cggtgtaggt cgttcgctcc aagctgggct
     1081 gtgtgcacga accccccgtt cagcccgacc gctgcgcctt atccggtaac tatcgtcttg
     1141 agtccaaccc ggtaagacac gacttatcgc cactggcagc agccactggt aacaggatta
     1201 gcagagcgag gtatgtaggc ggtgctacag agttcttgaa gtggtggcct aactacggct
     1261 acactagaag aacagtattt ggtatctgcg ctctgctgaa gccagttacc ttcggaaaaa
     1321 gagttggtag ctcttgatcc ggcaaacaaa ccaccgctgg tagcggtggt ttttttgttt
     1381 gcaagcagca gattacgcgc agaaaaaaag gatctcaaga agatcctttg atcttttcta
     1441 cggggtctga cgctcagtgg aacgaaaact cacgttaagg gattttggtc atgagattat
     1501 caaaaaggat cttcacctag atccttttaa attaaaaatg aagttttaaa tcaatctaaa
     1561 gtatatatga gtaaacttgg tctgacagtt accaatgctt aatcagtgag gcacctatct
     1621 cagcgatctg tctatttcgt tcatccatag ttgcctgact ccccgtcgtg tagataacta
     1681 cgatacggga gggcttacca tctggcccca gtgctgcaat gataccgcga gacccacgct
     1741 caccggctcc agatttatca gcaataaacc agccagccgg aagggccgag cgcagaagtg
     1801 gtcctgcaac tttatccgcc tccatccagt ctattaattg ttgccgggaa gctagagtaa
     1861 gtagttcgcc agttaatagt ttgcgcaacg ttgttgccat tgctacaggc atcgtggtgt
     1921 cacgctcgtc gtttggtatg gcttcattca gctccggttc ccaacgatca aggcgagtta
     1981 catgatcccc catgttgtgc aaaaaagcgg ttagctcctt cggtcctccg atcgttgtca
     2041 gaagtaagtt ggccgcagtg ttatcactca tggttatggc agcactgcat aattctctta
     2101 ctgtcatgcc atccgtaaga tgcttttctg tgactggtga gtactcaacc aagtcattct
     2161 gagaatagtg tatgcggcga ccgagttgct cttgcccggc gtcaatacgg gataataccg
     2221 cgccacatag cagaacttta aaagtgctca tcattggaaa acgttcttcg gggcgaaaac
     2281 tctcaaggat cttaccgctg ttgagatcca gttcgatgta acccactcgt gcacccaact
     2341 gatcttcagc atcttttact ttcaccagcg tttctgggtg agcaaaaaca ggaaggcaaa
     2401 atgccgcaaa aaagggaata agggcgacac ggaaatgttg aatactcata ctcttccttt
     2461 ttcaatatta ttgaagcatt tatcagggtt attgtctcat gagcggatac atatttgaat
     2521 gtatttagaa aaataaacaa ataggggttc cgcgcacatt tccccgaaaa gtgccacctg
     2581 ggtcgacatt gattattgat cgctagctgt acaaaaaagc aggctttaaa ggaaccaatt
     2641 cagtcgactg gatccggtac caaggtcggg caggaagagg gcctatttcc catgattcct
     2701 tcatatttgc atatacgata caaggctgtt agagagataa ttagaattaa tttgactgta
     2761 aacacaaaga tattagtaca aaatacgtga cgtagaaagt aataatttct tgggtagttt
     2821 gcagttttaa aattatgttt taaaatggac tatcatatgc ttaccgtaac ttgaaagtat
     2881 ttcgatttct tggctttata tatcttgtgg aaaggacgaa acaccgttca ctgccgtata
     2941 ggcagtgaga cgattaatgc gtctcagttt tagagctaga aatagcaagt taaaataagg
     3001 ctagtccgtt atcaacttga aaaagtggca ccgagtcggt gcgttcactg ccgtataggc
     3061 agttttttta agcttccgcg ttacataact tacggtaaat ggcccgcctg gctgaccgcc
     3121 caacgacccc cgcccattga cgtcaataat gacgtatgtt cccatagtaa cgccaatagg
     3181 gactttccat tgacgtcaat gggtggagta tttacggtaa actgcccact tggcagtaca
     3241 tcaagtgtat catatgccaa gtacgccccc tattgacgtc aatgacggta aatggcccgc
     3301 ctggcattat gcccagtaca tgaccttatg ggactttcct acttggcagt acatctacgt
     3361 attagtcatc gctattacca tggtgatgcg gttttggcag tacatcaatg ggcgtggata
     3421 gcggtttgac tcacggggat ttccaagtct ccaccccatt gacgtcaatg ggagtttgtt
     3481 ttggcaccaa aatcaacggg actttccaaa atgtcgtaac aactccgccc cattgacgca
     3541 aatgggcggt aggcgtgtac ggtgggaggt ctatataagc agagctcgtt tagtgaaccg
     3601 tcagatcgcc tggagtaccg cgggcccggg atccaccggt cgccaccatg ggtgatcatt
     3661 atctggatat tcggctgagg cctgatccag agttcccacc tgcgcagctg atgtctgtcc
     3721 tttttggcaa acttcatcag gccctggttg cccagggcgg agatcggata ggggtaagct
     3781 ttccagacct cgacgaaagc cggagccgcc tgggagaacg cctgcggatc cacgcttctg
     3841 ccgacgatct gagagccttg ctggcaaggc catggcttga ggggctccgg gatcacctgc
     3901 agtttggcga acccgccgtt gttccccacc caacccctta tcggcaggtg tctagagtgc
     3961 aggccaaatc taatccagaa cggctgcgac ggcgactcat gcggcgacat gatcttagcg
     4021 aggaagaggc ccgaaaaaga atccctgata ccgtggcccg cgcccttgac ttgccttttg
     4081 tcacactgcg gtcccagagt acggggcagc atttcagact tttcattcga cacgggccac
     4141 tgcaagttac cgccgaagaa ggaggcttta cttgttatgg actctccaag ggaggtttcg
     4201 tgccctggtt tgagggcaga ggaagtctgt taacatgcgg tgacgtcgag gagaatcctg
     4261 gcccaatgcc taagaagaag cggaaggtga gcagccaact tgtgaagtct gaactcgagg
     4321 agaaaaaatc agagttgaga cacaagttga agtacgtgcc acacgaatac atcgagctta
     4381 tcgagatcgc cagaaacagt acccaggata ggatccttga gatgaaagtc atggagttct
     4441 ttatgaaggt ctacggttat agaggaaagc accttggcgg tagcagaaag cccgatggcg
     4501 ccatctatac tgtcggatct cctatcgatt atggggtgat cgtggatacc aaagcttact
     4561 caggcgggta caacttgccc ataggacaag ccgacgagat gcagcggtat gtcgaagaga
     4621 accagacgcg caacaagcac atcaacccca atgaatggtg gaaagtgtac ccaagtagtg
     4681 tgactgagtt caagttcctg tttgtctccg gccactttaa gggcaattat aaagctcagc
     4741 tcactagact caatcacatc acaaactgca acggagctgt gttgtcagtg gaggagctcc
     4801 tgattggagg cgagatgatc aaagccggca cccttacact ggaggaggtg cggcggaagt
     4861 tcaacaatgg agagatcaac ttcggtggcg gtggatccgg tggcggtgga tccggtggcg
     4921 gtggatccgg tggcggtgga tccggtggtg gtggatccga taaaaagtat tctattggtt
     4981 tagccatcgg cactaattcc gttggatggg ctgtcataac cgatgaatac aaagtacctt
     5041 caaagaaatt taaggtgttg gggaacacag accgtcattc gattaaaaag aatcttatcg
     5101 gtgccctcct attcgatagt ggcgaaacgg cagaggcgac tcgcctgaaa cgaaccgctc
     5161 ggagaaggta tacacgtcgc aagaaccgaa tatgttactt acaagaaatt tttagcaatg
     5221 agatggccaa agttgacgat tctttctttc accgtttgga agagtccttc cttgtcgaag
     5281 aggacaagaa acatgaacgg caccccatct ttggaaacat agtagatgag gtggcatatc
     5341 atgaaaagta cccaacgatt tatcacctca gaaaaaagct agttgactca actgataaag
     5401 cggacctgag gttaatctac ttggctcttg cccatatgat aaagttccgt gggcactttc
     5461 tcattgaggg tgatctaaat ccggacaact cggatgtcga caaactgttc atccagttag
     5521 tacaaaccta taatcagttg tttgaagaga accctataaa tgcaagtggc gtggatgcga
     5581 aggctattct tagcgcccgc ctctctaaat cccgacggct agaaaacctg atcgcacaat
     5641 tacccggaga gaagaaaaat gggttgttcg gtaaccttat agcgctctca ctaggcctga
     5701 caccaaattt taagtcgaac ttcgacttag ctgaagatgc caaattgcag cttagtaagg
     5761 acacgtacga tgacgatctc gacaatctac tggcacaaat tggagatcag tatgcggact
     5821 tatttttggc tgccaaaaac cttagcgatg caatcctcct atctgacata ctgagagtta
     5881 atactgagat taccaaggcg ccgttatccg cttcaatgat caaaaggtac gatgaacatc
     5941 accaagactt gacacttctc aaggccctag tccgtcagca actgcctgag aaatataagg
     6001 aaatattctt tgatcagtcg aaaaacgggt acgcaggtta tattgacggc ggagcgagtc
     6061 aagaggaatt ctacaagttt atcaaaccca tattagagaa gatggatggg acggaagagt
     6121 tgcttgtaaa actcaatcgc gaagatctac tgcgaaagca gcggactttc gacaacggta
     6181 gcattccaca tcaaatccac ttaggcgaat tgcatgctat acttagaagg caggaggatt
     6241 tttatccgtt cctcaaagac aatcgtgaaa agattgagaa aatcctaacc tttcgcatac
     6301 cttactatgt gggacccctg gcccgaggga actctcggtt cgcatggatg acaagaaagt
     6361 ccgaagaaac gattactcca tggaattttg aggaagttgt cgataaaggt gcgtcagctc
     6421 aatcgttcat cgagaggatg accaactttg acaagaattt accgaacgaa aaagtattgc
     6481 ctaagcacag tttactttac gagtatttca cagtgtacaa tgaactcacg aaagttaagt
     6541 atgtcactga gggcatgcgt aaacccgcct ttctaagcgg agaacagaag aaagcaatag
     6601 tagatctgtt attcaagacc aaccgcaaag tgacagttaa gcaattgaaa gaggactact
     6661 ttaagaaaat tgaatgcttc gattctgtcg agatctccgg ggtagaagat cgatttaatg
     6721 cgtcacttgg tacgtatcat gacctcctaa agataattaa agataaggac ttcctggata
     6781 acgaagagaa tgaagatatc ttagaagata tagtgttgac tcttaccctc tttgaagatc
     6841 gggaaatgat tgaggaaaga ctaaaaacat acgctcacct gttcgacgat aaggttatga
     6901 aacagttaaa gaggcgtcgc tatacgggct ggggacgatt gtcgcggaaa cttatcaacg
     6961 ggataagaga caagcaaagt ggtaaaacta ttctcgattt tctaaagagc gacggcttcg
     7021 ccaataggaa ctttatgcag ctgatccatg atgactcttt aaccttcaaa gaggatatac
     7081 aaaaggcaca ggtttccgga caaggggact cattgcacga acatattgcg aatcttgctg
     7141 gttcgccagc catcaaaaag ggcatactcc agacagtcaa agtagtggat gagctagtta
     7201 aggtcatggg acgtcacaaa ccggaaaaca ttgtaatcga gatggcacgc gaaaatcaaa
     7261 cgactcagaa ggggcaaaaa aacagtcgag agcggatgaa gagaatagaa gagggtatta
     7321 aagaactggg cagccagatc ttaaaggagc atcctgtgga aaatacccaa ttgcagaacg
     7381 agaaacttta cctctattac ctacaaaatg gaagggacat gtatgttgat caggaactgg
     7441 acataaaccg tttatctgat tacgacgtcg atgccattgt accccaatcc tttttgaagg
     7501 acgattcaat cgacaataaa gtgcttacac gctcggataa gaaccgaggg aaaagtgaca
     7561 atgttccaag cgaggaagtc gtaaagaaaa tgaagaacta ttggcggcag ctcctaaatg
     7621 cgaaactgat aacgcaaaga aagttcgata acttaactaa agctgagagg ggtggcttgt
     7681 ctgaacttga caaggccgga tttattaaac gtcagctcgt ggaaacccgc caaatcacaa
     7741 agcatgttgc acagatacta gattcccgaa tgaatacgaa atacgacgag aacgataagc
     7801 tgattcggga agtcaaagta atcactttaa agtcaaaatt ggtgtcggac ttcagaaagg
     7861 attttcaatt ctataaagtt agggagataa ataactacca ccatgcgcac gacgcttatc
     7921 ttaatgccgt cgtagggacc gcactcatta agaaataccc gaagctagaa agtgagtttg
     7981 tgtatggtga ttacaaagtt tatgacgtcc gtaagatgat cgcgaaaagc gaacaggaga
     8041 taggcaaggc tacagccaaa tacttctttt attctaacat tatgaatttc tttaagacgg
     8101 aaatcactct ggcaaacgga gagatacgca aacgaccttt aattgaaacc aatggggaga
     8161 caggtgaaat cgtatgggat aagggccggg acttcgcgac ggtgagaaaa gttttgtcca
     8221 tgccccaagt caacatagta aagaaaactg aggtgcagac cggagggttt tcaaaggaat
     8281 cgattcttcc aaaaaggaat agtgataagc tcatcgctcg taaaaaggac tgggacccga
     8341 aaaagtacgg tggcttcgat agccctacag ttgcctattc tgtcctagta gtggcaaaag
     8401 ttgagaaggg aaaatccaag aaactgaagt cagtcaaaga attattgggg ataacgatta
     8461 tggagcgctc gtcttttgaa aagaacccca tcgacttcct tgaggcgaaa ggttacaagg
     8521 aagtaaaaaa ggatctcata attaaactac caaagtatag tctgtttgag ttagaaaatg
     8581 gccgaaaacg gatgttggct agcgccggag agcttcaaaa ggggaacgaa ctcgcactac
     8641 cgtctaaata cgtgaatttc ctgtatttag cgtcccatta cgagaagttg aaaggttcac
     8701 ctgaagataa cgaacagaag caactttttg ttgagcagca caaacattat ctcgacgaaa
     8761 tcatagagca aatttcggaa ttcagtaaga gagtcatcct agctgatgcc aatctggaca
     8821 aagtattaag cgcatacaac aagcacaggg ataaacccat acgtgagcag gcggaaaata
     8881 ttatccattt gtttactctt accaacctcg gcgctccagc cgcattcaag tattttgaca
     8941 caacgataga tcgcaaacga tacacttcta ccaaggaggt gctagacgcg acactgattc
     9001 accaatccat cacgggatta tatgaaactc ggatagattt gtcacagctt gggggtgacg
     9061 gatcccccaa gaagaagagg aaagtctcga gcgactacaa agaccatgac ggtgattata
     9121 aagatcatga catcgattac aaggatgacg atgacaaggg aagcggagcc actaacttct
     9181 ccctgttgaa acaagcaggg gatgtcgaag agaatcccgg gccagtgagc aagggcgagg
     9241 agctgttcac cggggtggtg cccatcctgg tcgagctgga cggcgacgta aacggccaca
     9301 agttcagcgt gtccggcgag ggcgagggcg atgccaccta cggcaagctg accctgaagt
     9361 tcatctgcac caccggcaag ctgcccgtgc cctggcccac cctcgtgacc accctgacct
     9421 acggcgtgca gtgcttcagc cgctaccccg accacatgaa gcagcacgac ttcttcaagt
     9481 ccgccatgcc cgaaggctac gtccaggagc gcaccatctt cttcaaggac gacggcaact
     9541 acaagacccg cgccgaggtg aagttcgagg gcgacaccct ggtgaaccgc atcgagctga
     9601 agggcatcga cttcaaggag gacggcaaca tcctggggca caagctggag tacaactaca
     9661 acagccacaa cgtctatatc atggccgaca agcagaagaa cggcatcaag gtgaacttca
     9721 agatccgcca caacatcgag gacggcagcg tgcagctcgc cgaccactac cagcagaaca
     9781 cccccatcgg cgacggcccc gtgctgctgc ccgacaacca ctacctgagc acccagtccg
     9841 ccctgagcaa agaccccaac gagaagcgcg atcacatggt cctgctggag ttcgtgaccg
     9901 ccgccgggat cactctcggc atggacgagc tgtacaagta aagcggccgc actcctcagg
     9961 tgcaggctgc ctatcagaag gtggtggctg gtgtggccaa tgccctggct cacaaatacc
    10021 actgagatct ttttccctct gccaaaaatt atggggacat catgaagccc cttgagcatc
    10081 tgacttctgg ctaataaagg aaatttattt tcattgcaat agtgtgttgg aattttttgt
    10141 gtctctcact cggaaggaca tatgggaggg caaatcattt aaaacatcag aatgagtatt
    10201 tggtttagag tttggcaaca tatgcccata tgctggctgc catgaacaaa ggttggctat
    10261 aaagaggtca tcagtatatg aaacagcccc ctgctgtcca ttccttattc catagaaaag
    10321 ccttgacttg aggttagatt ttttttatat tttgttttgt gttatttttt tctttaacat
    10381 ccctaaaatt ttccttacat gttttactag ccagattttt cctcct
//
