LOCUS       sequence-483540-        8506 bp    DNA     circular SYN 15-AUG-2025
DEFINITION  Cas9-gRNA construct targeting the UBE3A locus.
ACCESSION   .
VERSION     .
KEYWORDS    .
SOURCE      synthetic DNA construct
  ORGANISM  synthetic DNA construct
REFERENCE   1  (bases 1 to 8506)
  TITLE     Plasmids for UBE3A Reporter Stem cells
  JOURNAL   Unpublished
REFERENCE   2  (bases 1 to 8506)
  AUTHORS   .
  TITLE     Direct Submission
  JOURNAL   Exported Aug 15, 2025 from SnapGene Server 8.0.1
            https://www.snapgene.com
COMMENT     SGRef: number: 1; type: "Journal Article"; journalName: 
            "Unpublished"
COMMENT     Sequence Label: pDS48
FEATURES             Location/Qualifiers
     source          1..8506
                     /mol_type="other DNA"
                     /organism="synthetic DNA construct"
     promoter        6..254
                     /label=U6 promoter
                     /note="RNA polymerase III promoter for human U6 snRNA 
                     (Domitrovich & Kunkel, 2003)"
     primer_bind     6..26
                     /label=hU6-F
                     /note="Human U6 promoter, forward primer"
     primer_bind     177..196
                     /label=LKO.1 5'
                     /note="Human U6 promoter, forward primer"
     misc_RNA        274..349
                     /label=gRNA scaffold
                     /note="guide RNA scaffold for the Streptococcus pyogenes 
                     CRISPR/Cas9 system"
     enhancer        446..731
                     /label=CMV enhancer
                     /note="human cytomegalovirus immediate early enhancer; 
                     contains an 18-bp deletion relative to the standard CMV 
                     enhancer"
     promoter        733..1010
                     /label=chicken beta-actin promoter
     intron          1011..1238
                     /label=hybrid intron
                     /note="hybrid between chicken beta-actin (CBA) and minute 
                     virus of mice (MMV) introns (Gray et al., 2011)"
     regulatory      1250..1259
                     /label=Kozak sequence
                     /note="vertebrate consensus sequence for strong initiation 
                     of translation (Kozak, 1987)"
                     /regulatory_class="other"
     CDS             1259..1324
                     /codon_start=1
                     /product="three tandem FLAG(R) epitope tags, followed by an
                     enterokinase cleavage site"
                     /label=3xFLAG
                     /translation="DYKDHDGDYKDHDIDYKDDDDK"
     CDS             1331..1351
                     /codon_start=1
                     /product="nuclear localization signal of SV40 (simian virus
                     40) large T antigen"
                     /label=SV40 NLS
                     /translation="PKKKRKV"
     CDS             1376..5476
                     /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             1376..5476
                     /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             5477..5524
                     /codon_start=1
                     /product="bipartite nuclear localization signal from 
                     nucleoplasmin"
                     /label=nucleoplasmin NLS
                     /translation="KRPAATKKAGQAKKKK"
     primer_bind     complement(5552..5569)
                     /label=BGH-rev
                     /note="Bovine growth hormone terminator, reverse primer. 
                     Also called BGH reverse"
     polyA_signal    5558..5765
                     /label=bGH poly(A) signal
                     /note="bovine growth hormone polyadenylation signal"
     repeat_region   5774..5914
                     /label=AAV2 ITR
                     /note="inverted terminal repeat of adeno-associated virus 
                     serotype 2"
     repeat_region   5774..5903
                     /label=AAV2 ITR
     rep_origin      5989..6444
                     /direction=RIGHT
                     /label=f1 ori
                     /note="f1 bacteriophage origin of replication; arrow 
                     indicates direction of (+) strand synthesis"
     primer_bind     complement(6076..6095)
                     /label=F1ori-R
                     /note="F1 origin, reverse primer"
     primer_bind     6286..6307
                     /label=F1ori-F
                     /note="F1 origin, forward primer"
     primer_bind     complement(6461..6480)
                     /label=pRS-marker
                     /note="pRS vectors, use to sequence yeast selectable 
                     marker"
     primer_bind     6580..6602
                     /label=pGEX 3'
                     /note="pGEX vectors, reverse primer"
     primer_bind     complement(6640..6658)
                     /label=pBRforEco
                     /note="pBR322 vectors, upsteam of EcoRI site, forward 
                     primer"
     promoter        6726..6830
                     /gene="bla"
                     /label=AmpR promoter
     CDS             join(6831..6899,6900..7691)
                     /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(7049..7068)
                     /label=Amp-R
                     /note="Ampicillin resistance gene, reverse primer"
     rep_origin      7862..8450
                     /direction=RIGHT
                     /label=ori
                     /note="high-copy-number ColE1/pMB1/pBR322/pUC origin of 
                     replication"
     primer_bind     8351..8370
                     /label=pBR322ori-F
                     /note="pBR322 origin, forward primer"
ORIGIN
        1 catgtgaggg cctatttccc atgattcctt catatttgca tatacgatac aaggctgtta
       61 gagagataat tggaattaat ttgactgtaa acacaaagat attagtacaa aatacgtgac
      121 gtagaaagta ataatttctt gggtagtttg cagttttaaa attatgtttt aaaatggact
      181 atcatatgct taccgtaact tgaaagtatt tcgatttctt ggctttatat atcttgtgga
      241 aaggacgaaa caccaggcca tcacgtatgc caagttttag agctagaaat agcaagttaa
      301 aataaggcta gtccgttatc aacttgaaaa agtggcaccg agtcggtgct tttttgtttt
      361 agagctagaa atagcaagtt aaaataaggc tagtccgttt ttagcgcgtg cgccaattct
      421 gcagacaaat ggctctagag gtacccgtta cataacttac ggtaaatggc ccgcctggct
      481 gaccgcccaa cgacccccgc ccattgacgt caatagtaac gccaataggg actttccatt
      541 gacgtcaatg ggtggagtat ttacggtaaa ctgcccactt ggcagtacat caagtgtatc
      601 atatgccaag tacgccccct attgacgtca atgacggtaa atggcccgcc tggcattgtg
      661 cccagtacat gaccttatgg gactttccta cttggcagta catctacgta ttagtcatcg
      721 ctattaccat ggtcgaggtg agccccacgt tctgcttcac tctccccatc tcccccccct
      781 ccccaccccc aattttgtat ttatttattt tttaattatt ttgtgcagcg atgggggcgg
      841 gggggggggg ggggcgcgcg ccaggsgggg gggggggggg ggaggggsgg gggggggsga
      901 ggsggagagg tgsggcggca gccaatcaga gcggcgcgct ccgaaagttt ccttttatgg
      961 cgaggcggcg gcggcggcgg ccctataaaa agcgaagcgc gcggcgggcg ggagtcgctg
     1021 cgcgctgcct tcgccccgtg ccccgctccg ccgccgcctc gcgccgcccg ccccggctct
     1081 gactgaccgc gttactccca caggtgagcg ggcgggacgg cccttctcct ccgggctgta
     1141 attagctgag caagaggtaa gggtttaagg gatggttggt tggtggggta ttaatgttta
     1201 attacctgga gcacctgcct gaaatcactt tttttcaggt tggaccggtg ccaccatgga
     1261 ctataaggac cacgacggag actacaagga tcatgatatt gattacaaag acgatgacga
     1321 taagatggcc ccaaagaaga agcggaaggt cggtatccac ggagtcccag cagccgacaa
     1381 gaagtacagc atcggcctgg acatcggcac caactctgtg ggctgggccg tgatcaccga
     1441 cgagtacaag gtgcccagca agaaattcaa ggtgctgggc aacaccgacc ggcacagcat
     1501 caagaagaac ctgatcggag ccctgctgtt cgacagcggc gaaacagccg aggccacccg
     1561 gctgaagaga accgccagaa gaagatacac cagacggaag aaccggatct gctatctgca
     1621 agagatcttc agcaacgaga tggccaaggt ggacgacagc ttcttccaca gactggaaga
     1681 gtccttcctg gtggaagagg ataagaagca cgagcggcac cccatcttcg gcaacatcgt
     1741 ggacgaggtg gcctaccacg agaagtaccc caccatctac cacctgagaa agaaactggt
     1801 ggacagcacc gacaaggccg acctgcggct gatctatctg gccctggccc acatgatcaa
     1861 gttccggggc cacttcctga tcgagggcga cctgaacccc gacaacagcg acgtggacaa
     1921 gctgttcatc cagctggtgc agacctacaa ccagctgttc gaggaaaacc ccatcaacgc
     1981 cagcggcgtg gacgccaagg ccatcctgtc tgccagactg agcaagagca gacggctgga
     2041 aaatctgatc gcccagctgc ccggcgagaa gaagaatggc ctgttcggaa acctgattgc
     2101 cctgagcctg ggcctgaccc ccaacttcaa gagcaacttc gacctggccg aggatgccaa
     2161 actgcagctg agcaaggaca cctacgacga cgacctggac aacctgctgg cccagatcgg
     2221 cgaccagtac gccgacctgt ttctggccgc caagaacctg tccgacgcca tcctgctgag
     2281 cgacatcctg agagtgaaca ccgagatcac caaggccccc ctgagcgcct ctatgatcaa
     2341 gagatacgac gagcaccacc aggacctgac cctgctgaaa gctctcgtgc ggcagcagct
     2401 gcctgagaag tacaaagaga ttttcttcga ccagagcaag aacggctacg ccggctacat
     2461 tgacggcgga gccagccagg aagagttcta caagttcatc aagcccatcc tggaaaagat
     2521 ggacggcacc gaggaactgc tcgtgaagct gaacagagag gacctgctgc ggaagcagcg
     2581 gaccttcgac aacggcagca tcccccacca gatccacctg ggagagctgc acgccattct
     2641 gcggcggcag gaagattttt acccattcct gaaggacaac cgggaaaaga tcgagaagat
     2701 cctgaccttc cgcatcccct actacgtggg ccctctggcc aggggaaaca gcagattcgc
     2761 ctggatgacc agaaagagcg aggaaaccat caccccctgg aacttcgagg aagtggtgga
     2821 caagggcgct tccgcccaga gcttcatcga gcggatgacc aacttcgata agaacctgcc
     2881 caacgagaag gtgctgccca agcacagcct gctgtacgag tacttcaccg tgtataacga
     2941 gctgaccaaa gtgaaatacg tgaccgaggg aatgagaaag cccgccttcc tgagcggcga
     3001 gcagaaaaag gccatcgtgg acctgctgtt caagaccaac cggaaagtga ccgtgaagca
     3061 gctgaaagag gactacttca agaaaatcga gtgcttcgac tccgtggaaa tctccggcgt
     3121 ggaagatcgg ttcaacgcct ccctgggcac ataccacgat ctgctgaaaa ttatcaagga
     3181 caaggacttc ctggacaatg aggaaaacga ggacattctg gaagatatcg tgctgaccct
     3241 gacactgttt gaggacagag agatgatcga ggaacggctg aaaacctatg cccacctgtt
     3301 cgacgacaaa gtgatgaagc agctgaagcg gcggagatac accggctggg gcaggctgag
     3361 ccggaagctg atcaacggca tccgggacaa gcagtccggc aagacaatcc tggatttcct
     3421 gaagtccgac ggcttcgcca acagaaactt catgcagctg atccacgacg acagcctgac
     3481 ctttaaagag gacatccaga aagcccaggt gtccggccag ggcgatagcc tgcacgagca
     3541 cattgccaat ctggccggca gccccgccat taagaagggc atcctgcaga cagtgaaggt
     3601 ggtggacgag ctcgtgaaag tgatgggccg gcacaagccc gagaacatcg tgatcgaaat
     3661 ggccagagag aaccagacca cccagaaggg acagaagaac agccgcgaga gaatgaagcg
     3721 gatcgaagag ggcatcaaag agctgggcag ccagatcctg aaagaacacc ccgtggaaaa
     3781 cacccagctg cagaacgaga agctgtacct gtactacctg cagaatgggc gggatatgta
     3841 cgtggaccag gaactggaca tcaaccggct gtccgactac gatgtggacc atatcgtgcc
     3901 tcagagcttt ctgaaggacg actccatcga caacaaggtg ctgaccagaa gcgacaagaa
     3961 ccggggcaag agcgacaacg tgccctccga agaggtcgtg aagaagatga agaactactg
     4021 gcggcagctg ctgaacgcca agctgattac ccagagaaag ttcgacaatc tgaccaaggc
     4081 cgagagaggc ggcctgagcg aactggataa ggccggcttc atcaagagac agctggtgga
     4141 aacccggcag atcacaaagc acgtggcaca gatcctggac tcccggatga acactaagta
     4201 cgacgagaat gacaagctga tccgggaagt gaaagtgatc accctgaagt ccaagctggt
     4261 gtccgatttc cggaaggatt tccagtttta caaagtgcgc gagatcaaca actaccacca
     4321 cgcccacgac gcctacctga acgccgtcgt gggaaccgcc ctgatcaaaa agtaccctaa
     4381 gctggaaagc gagttcgtgt acggcgacta caaggtgtac gacgtgcgga agatgatcgc
     4441 caagagcgag caggaaatcg gcaaggctac cgccaagtac ttcttctaca gcaacatcat
     4501 gaactttttc aagaccgaga ttaccctggc caacggcgag atccggaagc ggcctctgat
     4561 cgagacaaac ggcgaaaccg gggagatcgt gtgggataag ggccgggatt ttgccaccgt
     4621 gcggaaagtg ctgagcatgc cccaagtgaa tatcgtgaaa aagaccgagg tgcagacagg
     4681 cggcttcagc aaagagtcta tcctgcccaa gaggaacagc gataagctga tcgccagaaa
     4741 gaaggactgg gaccctaaga agtacggcgg cttcgacagc cccaccgtgg cctattctgt
     4801 gctggtggtg gccaaagtgg aaaagggcaa gtccaagaaa ctgaagagtg tgaaagagct
     4861 gctggggatc accatcatgg aaagaagcag cttcgagaag aatcccatcg actttctgga
     4921 agccaagggc tacaaagaag tgaaaaagga cctgatcatc aagctgccta agtactccct
     4981 gttcgagctg gaaaacggcc ggaagagaat gctggcctct gccggcgaac tgcagaaggg
     5041 aaacgaactg gccctgccct ccaaatatgt gaacttcctg tacctggcca gccactatga
     5101 gaagctgaag ggctcccccg aggataatga gcagaaacag ctgtttgtgg aacagcacaa
     5161 gcactacctg gacgagatca tcgagcagat cagcgagttc tccaagagag tgatcctggc
     5221 cgacgctaat ctggacaaag tgctgtccgc ctacaacaag caccgggata agcccatcag
     5281 agagcaggcc gagaatatca tccacctgtt taccctgacc aatctgggag cccctgccgc
     5341 cttcaagtac tttgacacca ccatcgaccg gaagaggtac accagcacca aagaggtgct
     5401 ggacgccacc ctgatccacc agagcatcac cggcctgtac gagacacgga tcgacctgtc
     5461 tcagctggga ggcgacaaaa ggccggcggc cacgaaaaag gccggccagg caaaaaagaa
     5521 aaagtaagaa ttcctagagc tcgctgatca gcctcgactg tgccttctag ttgccagcca
     5581 tctgttgttt gcccctcccc cgtgccttcc ttgaccctgg aaggtgccac tcccactgtc
     5641 ctttcctaat aaaatgagga aattgcatcg cattgtctga gtaggtgtca ttctattctg
     5701 gggggtgggg tggggcagga cagcaagggg gaggattggg aagagaatag caggcatgct
     5761 ggggagcggc cgcaggaacc cctagtgatg gagttggcca ctccctctct gcgcgctcgc
     5821 tcgctcactg aggccgggcg accaaaggtc gcccgacgcc cgggctttgc ccgggcggcc
     5881 tcagtgagcg agcgagcgcg cagctgcctg caggggcgcc tgatgcggta ttttctcctt
     5941 acgcatctgt gcggtatttc acaccgcata cgtcaaagca accatagtac gcgccctgta
     6001 gcggcgcatt aagcgcggcg ggtgtggtgg ttacgcgcag cgtgaccgct acacttgcca
     6061 gcgccttagc gcccgctcct ttcgctttct tcccttcctt tctcgccacg ttcgccggct
     6121 ttccccgtca agctctaaat cgggggctcc ctttagggtt ccgatttagt gctttacggc
     6181 acctcgaccc caaaaaactt gatttgggtg atggttcacg tagtgggcca tcgccctgat
     6241 agacggtttt tcgccctttg acgttggagt ccacgttctt taatagtgga ctcttgttcc
     6301 aaactggaac aacactcaac tctatctcgg gctattcttt tgatttataa gggattttgc
     6361 cgatttcggt ctattggtta aaaaatgagc tgatttaaca aaaatttaac gcgaatttta
     6421 acaaaatatt aacgtttaca attttatggt gcactctcag tacaatctgc tctgatgccg
     6481 catagttaag ccagccccga cacccgccaa cacccgctga cgcgccctga cgggcttgtc
     6541 tgctcccggc atccgcttac agacaagctg tgaccgtctc cgggagctgc atgtgtcaga
     6601 ggttttcacc gtcatcaccg aaacgcgcga gacgaaaggg cctcgtgata cgcctatttt
     6661 tataggttaa tgtcatgata ataatggttt cttagacgtc aggtggcact tttcggggaa
     6721 atgtgcgcgg aacccctatt tgtttatttt tctaaataca ttcaaatatg tatccgctca
     6781 tgagacaata accctgataa atgcttcaat aatattgaaa aaggaagagt atgagtattc
     6841 aacatttccg tgtcgccctt attccctttt ttgcggcatt ttgccttcct gtttttgctc
     6901 acccagaaac gctggtgaaa gtaaaagatg ctgaagatca gttgggtgca cgagtgggtt
     6961 acatcgaact ggatctcaac agcggtaaga tccttgagag ttttcgcccc gaagaacgtt
     7021 ttccaatgat gagcactttt aaagttctgc tatgtggcgc ggtattatcc cgtattgacg
     7081 ccgggcaaga gcaactcggt cgccgcatac actattctca gaatgacttg gttgagtact
     7141 caccagtcac agaaaagcat cttacggatg gcatgacagt aagagaatta tgcagtgctg
     7201 ccataaccat gagtgataac actgcggcca acttacttct gacaacgatc ggaggaccga
     7261 aggagctaac cgcttttttg cacaacatgg gggatcatgt aactcgcctt gatcgttggg
     7321 aaccggagct gaatgaagcc ataccaaacg acgagcgtga caccacgatg cctgtagcaa
     7381 tggcaacaac gttgcgcaaa ctattaactg gcgaactact tactctagct tcccggcaac
     7441 aattaataga ctggatggag gcggataaag ttgcaggacc acttctgcgc tcggcccttc
     7501 cggctggctg gtttattgct gataaatctg gagccggtga gcgtggaagc cgcggtatca
     7561 ttgcagcact ggggccagat ggtaagccct cccgtatcgt agttatctac acgacgggga
     7621 gtcaggcaac tatggatgaa cgaaatagac agatcgctga gataggtgcc tcactgatta
     7681 agcattggta actgtcagac caagtttact catatatact ttagattgat ttaaaacttc
     7741 atttttaatt taaaaggatc taggtgaaga tcctttttga taatctcatg accaaaatcc
     7801 cttaacgtga gttttcgttc cactgagcgt cagaccccgt agaaaagatc aaaggatctt
     7861 cttgagatcc tttttttctg cgcgtaatct gctgcttgca aacaaaaaaa ccaccgctac
     7921 cagcggtggt ttgtttgccg gatcaagagc taccaactct ttttccgaag gtaactggct
     7981 tcagcagagc gcagatacca aatactgttc ttctagtgta gccgtagtta ggccaccact
     8041 tcaagaactc tgtagcaccg cctacatacc tcgctctgct aatcctgtta ccagtggctg
     8101 ctgccagtgg cgataagtcg tgtcttaccg ggttggactc aagacgatag ttaccggata
     8161 aggcgcagcg gtcgggctga acggggggtt cgtgcacaca gcccagcttg gagcgaacga
     8221 cctacaccga actgagatac ctacagcgtg agctatgaga aagcgccacg cttcccgaag
     8281 ggagaaaggc ggacaggtat ccggtaagcg gcagggtcgg aacaggagag cgcacgaggg
     8341 agcttccagg gggaaacgcc tggtatcttt atagtcctgt cgggtttcgc cacctctgac
     8401 ttgagcgtcg atttttgtga tgctcgtcag gggggcggag cctatggaaa aacgccagca
     8461 acgcggcctt tttacggttc ctggcctttt gctggccttt tgctca
//
