LOCUS       sequence-369478-        8447 bp    DNA     circular SYN 15-DEC-2025
DEFINITION  A human codon-optimized SpCas9 and chimeric guide RNA targeting 
            C-terminus of human EGFR.
ACCESSION   .
VERSION     .
KEYWORDS    .
SOURCE      synthetic DNA construct
  ORGANISM  synthetic DNA construct
REFERENCE   1  (bases 1 to 8447)
  AUTHORS   Farahani PE, Yang X, Mesev EV, Fomby KA, Brumbaugh-Reed EH, Bashor 
            CJ, Nelson CM, Toettcher JE
  TITLE     pYtags enable spatiotemporal measurements of receptor tyrosine 
            kinase signaling in living cells.
  JOURNAL   eLife. 2023 May 22;12:e82863. doi: 10.7554/eLife.82863.
  PUBMED    37212240
REFERENCE   2  (bases 1 to 8447)
  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: "eLife."; 
            date: "2023-05-22"; volume: "12:e82863. doi"; pages: " 
            10.7554/eLife.82863"
COMMENT     Sequence Label: pX330 EGFR-sgRNA
FEATURES             Location/Qualifiers
     source          1..8447
                     /mol_type="other DNA"
                     /organism="synthetic DNA construct"
     intron          103..330
                     /label=hybrid intron
                     /note="hybrid between chicken beta-actin (CBA) and minute 
                     virus of mice (MMV) introns (Gray et al., 2011)"
     regulatory      342..351
                     /label=Kozak sequence
                     /note="vertebrate consensus sequence for strong initiation 
                     of translation (Kozak, 1987)"
                     /regulatory_class="other"
     CDS             351..416
                     /codon_start=1
                     /product="three tandem FLAG(R) epitope tags, followed by an
                     enterokinase cleavage site"
                     /label=3xFLAG
                     /translation="DYKDHDGDYKDHDIDYKDDDDK"
     CDS             423..443
                     /codon_start=1
                     /product="nuclear localization signal of SV40 (simian virus
                     40) large T antigen"
                     /label=SV40 NLS
                     /translation="PKKKRKV"
     CDS             468..4568
                     /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             468..4568
                     /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             4569..4616
                     /codon_start=1
                     /product="bipartite nuclear localization signal from 
                     nucleoplasmin"
                     /label=nucleoplasmin NLS
                     /translation="KRPAATKKAGQAKKKK"
     primer_bind     complement(4644..4661)
                     /label=BGH-rev
                     /note="Bovine growth hormone terminator, reverse primer. 
                     Also called BGH reverse"
     polyA_signal    4650..4857
                     /label=bGH poly(A) signal
                     /note="bovine growth hormone polyadenylation signal"
     repeat_region   4866..5006
                     /label=AAV2 ITR
                     /note="inverted terminal repeat of adeno-associated virus 
                     serotype 2"
     repeat_region   4866..4995
                     /label=AAV2 ITR
     rep_origin      5081..5536
                     /direction=RIGHT
                     /label=f1 ori
                     /note="f1 bacteriophage origin of replication; arrow 
                     indicates direction of (+) strand synthesis"
     primer_bind     complement(5168..5187)
                     /label=F1ori-R
                     /note="F1 origin, reverse primer"
     primer_bind     5378..5399
                     /label=F1ori-F
                     /note="F1 origin, forward primer"
     primer_bind     complement(5553..5572)
                     /label=pRS-marker
                     /note="pRS vectors, use to sequence yeast selectable 
                     marker"
     primer_bind     5672..5694
                     /label=pGEX 3'
                     /note="pGEX vectors, reverse primer"
     primer_bind     complement(5732..5750)
                     /label=pBRforEco
                     /note="pBR322 vectors, upsteam of EcoRI site, forward 
                     primer"
     promoter        5818..5922
                     /gene="bla"
                     /label=AmpR promoter
     CDS             join(5923..5991,5992..6783)
                     /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(6141..6160)
                     /label=Amp-R
                     /note="Ampicillin resistance gene, reverse primer"
     rep_origin      6954..7542
                     /direction=RIGHT
                     /label=ori
                     /note="high-copy-number ColE1/pMB1/pBR322/pUC origin of 
                     replication"
     primer_bind     7443..7462
                     /label=pBR322ori-F
                     /note="pBR322 origin, forward primer"
     promoter        7604..7852
                     /label=U6 promoter
                     /note="RNA polymerase III promoter for human U6 snRNA 
                     (Domitrovich & Kunkel, 2003)"
     primer_bind     7604..7624
                     /label=hU6-F
                     /note="Human U6 promoter, forward primer"
     primer_bind     7775..7794
                     /label=LKO.1 5'
                     /note="Human U6 promoter, forward primer"
     misc_RNA        7874..7949
                     /label=gRNA scaffold
                     /note="guide RNA scaffold for the Streptococcus pyogenes 
                     CRISPR/Cas9 system"
     enhancer        8046..8331
                     /label=CMV enhancer
                     /note="human cytomegalovirus immediate early enhancer; 
                     contains an 18-bp deletion relative to the standard CMV 
                     enhancer"
ORIGIN
        1 ggtgcggcgg cagccaatca gagcggcgcg ctccgaaagt ttccttttat ggcgaggcgg
       61 cggcggcggc ggccctataa aaagcgaagc gcgcggcggg cgggagtcgc tgcgcgctgc
      121 cttcgccccg tgccccgctc cgccgccgcc tcgcgccgcc cgccccggct ctgactgacc
      181 gcgttactcc cacaggtgag cgggcgggac ggcccttctc ctccgggctg taattagctg
      241 agcaagaggt aagggtttaa gggatggttg gttggtgggg tattaatgtt taattacctg
      301 gagcacctgc ctgaaatcac tttttttcag gttggaccgg tgccaccatg gactataagg
      361 accacgacgg agactacaag gatcatgata ttgattacaa agacgatgac gataagatgg
      421 ccccaaagaa gaagcggaag gtcggtatcc acggagtccc agcagccgac aagaagtaca
      481 gcatcggcct ggacatcggc accaactctg tgggctgggc cgtgatcacc gacgagtaca
      541 aggtgcccag caagaaattc aaggtgctgg gcaacaccga ccggcacagc atcaagaaga
      601 acctgatcgg agccctgctg ttcgacagcg gcgaaacagc cgaggccacc cggctgaaga
      661 gaaccgccag aagaagatac accagacgga agaaccggat ctgctatctg caagagatct
      721 tcagcaacga gatggccaag gtggacgaca gcttcttcca cagactggaa gagtccttcc
      781 tggtggaaga ggataagaag cacgagcggc accccatctt cggcaacatc gtggacgagg
      841 tggcctacca cgagaagtac cccaccatct accacctgag aaagaaactg gtggacagca
      901 ccgacaaggc cgacctgcgg ctgatctatc tggccctggc ccacatgatc aagttccggg
      961 gccacttcct gatcgagggc gacctgaacc ccgacaacag cgacgtggac aagctgttca
     1021 tccagctggt gcagacctac aaccagctgt tcgaggaaaa ccccatcaac gccagcggcg
     1081 tggacgccaa ggccatcctg tctgccagac tgagcaagag cagacggctg gaaaatctga
     1141 tcgcccagct gcccggcgag aagaagaatg gcctgttcgg aaacctgatt gccctgagcc
     1201 tgggcctgac ccccaacttc aagagcaact tcgacctggc cgaggatgcc aaactgcagc
     1261 tgagcaagga cacctacgac gacgacctgg acaacctgct ggcccagatc ggcgaccagt
     1321 acgccgacct gtttctggcc gccaagaacc tgtccgacgc catcctgctg agcgacatcc
     1381 tgagagtgaa caccgagatc accaaggccc ccctgagcgc ctctatgatc aagagatacg
     1441 acgagcacca ccaggacctg accctgctga aagctctcgt gcggcagcag ctgcctgaga
     1501 agtacaaaga gattttcttc gaccagagca agaacggcta cgccggctac attgacggcg
     1561 gagccagcca ggaagagttc tacaagttca tcaagcccat cctggaaaag atggacggca
     1621 ccgaggaact gctcgtgaag ctgaacagag aggacctgct gcggaagcag cggaccttcg
     1681 acaacggcag catcccccac cagatccacc tgggagagct gcacgccatt ctgcggcggc
     1741 aggaagattt ttacccattc ctgaaggaca accgggaaaa gatcgagaag atcctgacct
     1801 tccgcatccc ctactacgtg ggccctctgg ccaggggaaa cagcagattc gcctggatga
     1861 ccagaaagag cgaggaaacc atcaccccct ggaacttcga ggaagtggtg gacaagggcg
     1921 cttccgccca gagcttcatc gagcggatga ccaacttcga taagaacctg cccaacgaga
     1981 aggtgctgcc caagcacagc ctgctgtacg agtacttcac cgtgtataac gagctgacca
     2041 aagtgaaata cgtgaccgag ggaatgagaa agcccgcctt cctgagcggc gagcagaaaa
     2101 aggccatcgt ggacctgctg ttcaagacca accggaaagt gaccgtgaag cagctgaaag
     2161 aggactactt caagaaaatc gagtgcttcg actccgtgga aatctccggc gtggaagatc
     2221 ggttcaacgc ctccctgggc acataccacg atctgctgaa aattatcaag gacaaggact
     2281 tcctggacaa tgaggaaaac gaggacattc tggaagatat cgtgctgacc ctgacactgt
     2341 ttgaggacag agagatgatc gaggaacggc tgaaaaccta tgcccacctg ttcgacgaca
     2401 aagtgatgaa gcagctgaag cggcggagat acaccggctg gggcaggctg agccggaagc
     2461 tgatcaacgg catccgggac aagcagtccg gcaagacaat cctggatttc ctgaagtccg
     2521 acggcttcgc caacagaaac ttcatgcagc tgatccacga cgacagcctg acctttaaag
     2581 aggacatcca gaaagcccag gtgtccggcc agggcgatag cctgcacgag cacattgcca
     2641 atctggccgg cagccccgcc attaagaagg gcatcctgca gacagtgaag gtggtggacg
     2701 agctcgtgaa agtgatgggc cggcacaagc ccgagaacat cgtgatcgaa atggccagag
     2761 agaaccagac cacccagaag ggacagaaga acagccgcga gagaatgaag cggatcgaag
     2821 agggcatcaa agagctgggc agccagatcc tgaaagaaca ccccgtggaa aacacccagc
     2881 tgcagaacga gaagctgtac ctgtactacc tgcagaatgg gcgggatatg tacgtggacc
     2941 aggaactgga catcaaccgg ctgtccgact acgatgtgga ccatatcgtg cctcagagct
     3001 ttctgaagga cgactccatc gacaacaagg tgctgaccag aagcgacaag aaccggggca
     3061 agagcgacaa cgtgccctcc gaagaggtcg tgaagaagat gaagaactac tggcggcagc
     3121 tgctgaacgc caagctgatt acccagagaa agttcgacaa tctgaccaag gccgagagag
     3181 gcggcctgag cgaactggat aaggccggct tcatcaagag acagctggtg gaaacccggc
     3241 agatcacaaa gcacgtggca cagatcctgg actcccggat gaacactaag tacgacgaga
     3301 atgacaagct gatccgggaa gtgaaagtga tcaccctgaa gtccaagctg gtgtccgatt
     3361 tccggaagga tttccagttt tacaaagtgc gcgagatcaa caactaccac cacgcccacg
     3421 acgcctacct gaacgccgtc gtgggaaccg ccctgatcaa aaagtaccct aagctggaaa
     3481 gcgagttcgt gtacggcgac tacaaggtgt acgacgtgcg gaagatgatc gccaagagcg
     3541 agcaggaaat cggcaaggct accgccaagt acttcttcta cagcaacatc atgaactttt
     3601 tcaagaccga gattaccctg gccaacggcg agatccggaa gcggcctctg atcgagacaa
     3661 acggcgaaac cggggagatc gtgtgggata agggccggga ttttgccacc gtgcggaaag
     3721 tgctgagcat gccccaagtg aatatcgtga aaaagaccga ggtgcagaca ggcggcttca
     3781 gcaaagagtc tatcctgccc aagaggaaca gcgataagct gatcgccaga aagaaggact
     3841 gggaccctaa gaagtacggc ggcttcgaca gccccaccgt ggcctattct gtgctggtgg
     3901 tggccaaagt ggaaaagggc aagtccaaga aactgaagag tgtgaaagag ctgctgggga
     3961 tcaccatcat ggaaagaagc agcttcgaga agaatcccat cgactttctg gaagccaagg
     4021 gctacaaaga agtgaaaaag gacctgatca tcaagctgcc taagtactcc ctgttcgagc
     4081 tggaaaacgg ccggaagaga atgctggcct ctgccggcga actgcagaag ggaaacgaac
     4141 tggccctgcc ctccaaatat gtgaacttcc tgtacctggc cagccactat gagaagctga
     4201 agggctcccc cgaggataat gagcagaaac agctgtttgt ggaacagcac aagcactacc
     4261 tggacgagat catcgagcag atcagcgagt tctccaagag agtgatcctg gccgacgcta
     4321 atctggacaa agtgctgtcc gcctacaaca agcaccggga taagcccatc agagagcagg
     4381 ccgagaatat catccacctg tttaccctga ccaatctggg agcccctgcc gccttcaagt
     4441 actttgacac caccatcgac cggaagaggt acaccagcac caaagaggtg ctggacgcca
     4501 ccctgatcca ccagagcatc accggcctgt acgagacacg gatcgacctg tctcagctgg
     4561 gaggcgacaa aaggccggcg gccacgaaaa aggccggcca ggcaaaaaag aaaaagtaag
     4621 aattcctaga gctcgctgat cagcctcgac tgtgccttct agttgccagc catctgttgt
     4681 ttgcccctcc cccgtgcctt ccttgaccct ggaaggtgcc actcccactg tcctttccta
     4741 ataaaatgag gaaattgcat cgcattgtct gagtaggtgt cattctattc tggggggtgg
     4801 ggtggggcag gacagcaagg gggaggattg ggaagagaat agcaggcatg ctggggagcg
     4861 gccgcaggaa cccctagtga tggagttggc cactccctct ctgcgcgctc gctcgctcac
     4921 tgaggccggg cgaccaaagg tcgcccgacg cccgggcttt gcccgggcgg cctcagtgag
     4981 cgagcgagcg cgcagctgcc tgcaggggcg cctgatgcgg tattttctcc ttacgcatct
     5041 gtgcggtatt tcacaccgca tacgtcaaag caaccatagt acgcgccctg tagcggcgca
     5101 ttaagcgcgg cgggtgtggt ggttacgcgc agcgtgaccg ctacacttgc cagcgcctta
     5161 gcgcccgctc ctttcgcttt cttcccttcc tttctcgcca cgttcgccgg ctttccccgt
     5221 caagctctaa atcgggggct ccctttaggg ttccgattta gtgctttacg gcacctcgac
     5281 cccaaaaaac ttgatttggg tgatggttca cgtagtgggc catcgccctg atagacggtt
     5341 tttcgccctt tgacgttgga gtccacgttc tttaatagtg gactcttgtt ccaaactgga
     5401 acaacactca actctatctc gggctattct tttgatttat aagggatttt gccgatttcg
     5461 gtctattggt taaaaaatga gctgatttaa caaaaattta acgcgaattt taacaaaata
     5521 ttaacgttta caattttatg gtgcactctc agtacaatct gctctgatgc cgcatagtta
     5581 agccagcccc gacacccgcc aacacccgct gacgcgccct gacgggcttg tctgctcccg
     5641 gcatccgctt acagacaagc tgtgaccgtc tccgggagct gcatgtgtca gaggttttca
     5701 ccgtcatcac cgaaacgcgc gagacgaaag ggcctcgtga tacgcctatt tttataggtt
     5761 aatgtcatga taataatggt ttcttagacg tcaggtggca cttttcgggg aaatgtgcgc
     5821 ggaaccccta tttgtttatt tttctaaata cattcaaata tgtatccgct catgagacaa
     5881 taaccctgat aaatgcttca ataatattga aaaaggaaga gtatgagtat tcaacatttc
     5941 cgtgtcgccc ttattccctt ttttgcggca ttttgccttc ctgtttttgc tcacccagaa
     6001 acgctggtga aagtaaaaga tgctgaagat cagttgggtg cacgagtggg ttacatcgaa
     6061 ctggatctca acagcggtaa gatccttgag agttttcgcc ccgaagaacg ttttccaatg
     6121 atgagcactt ttaaagttct gctatgtggc gcggtattat cccgtattga cgccgggcaa
     6181 gagcaactcg gtcgccgcat acactattct cagaatgact tggttgagta ctcaccagtc
     6241 acagaaaagc atcttacgga tggcatgaca gtaagagaat tatgcagtgc tgccataacc
     6301 atgagtgata acactgcggc caacttactt ctgacaacga tcggaggacc gaaggagcta
     6361 accgcttttt tgcacaacat gggggatcat gtaactcgcc ttgatcgttg ggaaccggag
     6421 ctgaatgaag ccataccaaa cgacgagcgt gacaccacga tgcctgtagc aatggcaaca
     6481 acgttgcgca aactattaac tggcgaacta cttactctag cttcccggca acaattaata
     6541 gactggatgg aggcggataa agttgcagga ccacttctgc gctcggccct tccggctggc
     6601 tggtttattg ctgataaatc tggagccggt gagcgtggaa gccgcggtat cattgcagca
     6661 ctggggccag atggtaagcc ctcccgtatc gtagttatct acacgacggg gagtcaggca
     6721 actatggatg aacgaaatag acagatcgct gagataggtg cctcactgat taagcattgg
     6781 taactgtcag accaagttta ctcatatata ctttagattg atttaaaact tcatttttaa
     6841 tttaaaagga tctaggtgaa gatccttttt gataatctca tgaccaaaat cccttaacgt
     6901 gagttttcgt tccactgagc gtcagacccc gtagaaaaga tcaaaggatc ttcttgagat
     6961 cctttttttc tgcgcgtaat ctgctgcttg caaacaaaaa aaccaccgct accagcggtg
     7021 gtttgtttgc cggatcaaga gctaccaact ctttttccga aggtaactgg cttcagcaga
     7081 gcgcagatac caaatactgt tcttctagtg tagccgtagt taggccacca cttcaagaac
     7141 tctgtagcac cgcctacata cctcgctctg ctaatcctgt taccagtggc tgctgccagt
     7201 ggcgataagt cgtgtcttac cgggttggac tcaagacgat agttaccgga taaggcgcag
     7261 cggtcgggct gaacgggggg ttcgtgcaca cagcccagct tggagcgaac gacctacacc
     7321 gaactgagat acctacagcg tgagctatga gaaagcgcca cgcttcccga agggagaaag
     7381 gcggacaggt atccggtaag cggcagggtc ggaacaggag agcgcacgag ggagcttcca
     7441 gggggaaacg cctggtatct ttatagtcct gtcgggtttc gccacctctg acttgagcgt
     7501 cgatttttgt gatgctcgtc aggggggcgg agcctatgga aaaacgccag caacgcggcc
     7561 tttttacggt tcctggcctt ttgctggcct tttgctcaca tgtgagggcc tatttcccat
     7621 gattccttca tatttgcata tacgatacaa ggctgttaga gagataattg gaattaattt
     7681 gactgtaaac acaaagatat tagtacaaaa tacgtgacgt agaaagtaat aatttcttgg
     7741 gtagtttgca gttttaaaat tatgttttaa aatggactat catatgctta ccgtaacttg
     7801 aaagtatttc gatttcttgg ctttatatat cttgtggaaa ggacgaaaca ccgtccagac
     7861 tctttcgata cccgttttag agctagaaat agcaagttaa aataaggcta gtccgttatc
     7921 aacttgaaaa agtggcaccg agtcggtgct tttttgtttt agagctagaa atagcaagtt
     7981 aaaataaggc tagtccgttt ttagcgcgtg cgccaattct gcagacaaat ggctctagag
     8041 gtacccgtta cataacttac ggtaaatggc ccgcctggct gaccgcccaa cgacccccgc
     8101 ccattgacgt caatagtaac gccaataggg actttccatt gacgtcaatg ggtggagtat
     8161 ttacggtaaa ctgcccactt ggcagtacat caagtgtatc atatgccaag tacgccccct
     8221 attgacgtca atgacggtaa atggcccgcc tggcattgtg cccagtacat gaccttatgg
     8281 gactttccta cttggcagta catctacgta ttagtcatcg ctattaccat ggtcgaggtg
     8341 agccccacgt tctgcttcac tctccccatc tcccccccct ccccaccccc aattttgtat
     8401 ttatttattt tttaattatt ttgtgcagcg atgggggcgg ggggggg
//
