LOCUS       sequence-451733-        8950 bp    DNA     circular SYN 15-DEC-2025
DEFINITION  Expresses SpCas9, the PITCh gRNA, and a sgRNA targeting the 
            C-terminus of VIM for knock-in..
ACCESSION   .
VERSION     .
KEYWORDS    .
SOURCE      synthetic DNA construct
  ORGANISM  synthetic DNA construct
REFERENCE   1  (bases 1 to 8950)
  AUTHORS   Philip R, Sharma A, Matellan L, Erpf AC, Hsu WH, Tkach JM, Wyatt 
            HDM, Pelletier L
  TITLE     qTAG: an adaptable plasmid scaffold for CRISPR-based endogenous 
            tagging.
  JOURNAL   EMBO J. 2024 Dec 12. doi: 10.1038/s44318-024-00337-5.
  PUBMED    39668248
REFERENCE   2  (bases 1 to 8950)
  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: "EMBO J. 
            2024 Dec 12. doi: 10.1038/s44318-024-00337-5."
COMMENT     Sequence Label: pX330-PITCh-VIM
FEATURES             Location/Qualifiers
     source          1..8950
                     /mol_type="other DNA"
                     /organism="synthetic DNA construct"
     rep_origin      40..628
                     /direction=RIGHT
                     /label=ori
                     /note="high-copy-number ColE1/pMB1/pBR322/pUC origin of 
                     replication"
     primer_bind     529..548
                     /label=pBR322ori-F
                     /note="pBR322 origin, forward primer"
     promoter        690..938
                     /label=U6 promoter
                     /note="RNA polymerase III promoter for human U6 snRNA 
                     (Domitrovich & Kunkel, 2003)"
     misc_RNA        960..1035
                     /label=gRNA scaffold
                     /note="guide RNA scaffold for the Streptococcus pyogenes 
                     CRISPR/Cas9 system"
     promoter        1133..1381
                     /label=U6 promoter
                     /note="RNA polymerase III promoter for human U6 snRNA 
                     (Domitrovich & Kunkel, 2003)"
     misc_RNA        1402..1477
                     /label=gRNA scaffold
                     /note="guide RNA scaffold for the Streptococcus pyogenes 
                     CRISPR/Cas9 system"
     enhancer        1574..1859
                     /label=CMV enhancer
                     /note="human cytomegalovirus immediate early enhancer; 
                     contains an 18-bp deletion relative to the standard CMV 
                     enhancer"
     intron          2139..2366
                     /label=hybrid intron
                     /note="hybrid between chicken beta-actin (CBA) and minute 
                     virus of mice (MMV) introns (Gray et al., 2011)"
     regulatory      2378..2387
                     /label=Kozak sequence
                     /note="vertebrate consensus sequence for strong initiation 
                     of translation (Kozak, 1987)"
                     /regulatory_class="other"
     CDS             2387..2452
                     /codon_start=1
                     /product="three tandem FLAG(R) epitope tags, followed by an
                     enterokinase cleavage site"
                     /label=3xFLAG
                     /translation="DYKDHDGDYKDHDIDYKDDDDK"
     CDS             2459..2479
                     /codon_start=1
                     /product="nuclear localization signal of SV40 (simian virus
                     40) large T antigen"
                     /label=SV40 NLS
                     /translation="PKKKRKV"
     CDS             2504..6604
                     /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             2504..6604
                     /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             6605..6652
                     /codon_start=1
                     /product="bipartite nuclear localization signal from 
                     nucleoplasmin"
                     /label=nucleoplasmin NLS
                     /translation="KRPAATKKAGQAKKKK"
     primer_bind     complement(6680..6697)
                     /label=BGH-rev
                     /note="Bovine growth hormone terminator, reverse primer. 
                     Also called BGH reverse"
     polyA_signal    6686..6893
                     /label=bGH poly(A) signal
                     /note="bovine growth hormone polyadenylation signal"
     repeat_region   6902..7042
                     /label=AAV2 ITR
                     /note="inverted terminal repeat of adeno-associated virus 
                     serotype 2"
     repeat_region   6902..7031
                     /label=AAV2 ITR
     rep_origin      7117..7572
                     /direction=RIGHT
                     /label=f1 ori
                     /note="f1 bacteriophage origin of replication; arrow 
                     indicates direction of (+) strand synthesis"
     primer_bind     complement(7204..7223)
                     /label=F1ori-R
                     /note="F1 origin, reverse primer"
     primer_bind     7414..7435
                     /label=F1ori-F
                     /note="F1 origin, forward primer"
     primer_bind     complement(7589..7608)
                     /label=pRS-marker
                     /note="pRS vectors, use to sequence yeast selectable 
                     marker"
     primer_bind     7708..7730
                     /label=pGEX 3'
                     /note="pGEX vectors, reverse primer"
     primer_bind     complement(7768..7786)
                     /label=pBRforEco
                     /note="pBR322 vectors, upsteam of EcoRI site, forward 
                     primer"
     promoter        7854..7958
                     /gene="bla"
                     /label=AmpR promoter
     CDS             join(7959..8027,8028..8819)
                     /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(8177..8196)
                     /label=Amp-R
                     /note="Ampicillin resistance gene, reverse primer"
ORIGIN
        1 ctgagcgtca gaccccgtag aaaagatcaa aggatcttct tgagatcctt tttttctgcg
       61 cgtaatctgc tgcttgcaaa caaaaaaacc accgctacca gcggtggttt gtttgccgga
      121 tcaagagcta ccaactcttt ttccgaaggt aactggcttc agcagagcgc agataccaaa
      181 tactgttctt ctagtgtagc cgtagttagg ccaccacttc aagaactctg tagcaccgcc
      241 tacatacctc gctctgctaa tcctgttacc agtggctgct gccagtggcg ataagtcgtg
      301 tcttaccggg ttggactcaa gacgatagtt accggataag gcgcagcggt cgggctgaac
      361 ggggggttcg tgcacacagc ccagcttgga gcgaacgacc tacaccgaac tgagatacct
      421 acagcgtgag ctatgagaaa gcgccacgct tcccgaaggg agaaaggcgg acaggtatcc
      481 ggtaagcggc agggtcggaa caggagagcg cacgagggag cttccagggg gaaacgcctg
      541 gtatctttat agtcctgtcg ggtttcgcca cctctgactt gagcgtcgat ttttgtgatg
      601 ctcgtcaggg gggcggagcc tatggaaaaa cgccagcaac gcggcctttt tacggttcct
      661 ggccttttgc tggccttttg ctcacatgtg agggcctatt tcccatgatt ccttcatatt
      721 tgcatatacg atacaaggct gttagagaga taattggaat taatttgact gtaaacacaa
      781 agatattagt acaaaatacg tgacgtagaa agtaataatt tcttgggtag tttgcagttt
      841 taaaattatg ttttaaaatg gactatcata tgcttaccgt aacttgaaag tatttcgatt
      901 tcttggcttt atatatcttg tggaaaggac gaaacaccgg cgcaagatag atttggaatg
      961 ttttagagct agaaatagca agttaaaata aggctagtcc gttatcaact tgaaaaagtg
     1021 gcaccgagtc ggtgcttttt tgttttagag ctagaaatag caagttaaaa taaggctagt
     1081 ccgtttttag cgcgtgcgcc aattctgcag acaaatggct ctagaggcat gtgagggcct
     1141 atttcccatg attccttcat atttgcatat acgatacaag gctgttagag agataattgg
     1201 aattaatttg actgtaaaca caaagatatt agtacaaaat acgtgacgta gaaagtaata
     1261 atttcttggg tagtttgcag ttttaaaatt atgttttaaa atggactatc atatgcttac
     1321 cgtaacttga aagtatttcg atttcttggc tttatatatc ttgtggaaag gacgaaacac
     1381 cgcatcgtac gcgtacgtgt tgttttagag ctagaaatag caagttaaaa taaggctagt
     1441 ccgttatcaa cttgaaaaag tggcaccgag tcggtgcttt tttgttttag agctagaaat
     1501 agcaagttaa aataaggcta gtccgttttt agcgcgtgcg ccaattctgc agacaaatgg
     1561 ctctagaggt acccgttaca taacttacgg taaatggccc gcctggctga ccgcccaacg
     1621 acccccgccc attgacgtca atagtaacgc caatagggac tttccattga cgtcaatggg
     1681 tggagtattt acggtaaact gcccacttgg cagtacatca agtgtatcat atgccaagta
     1741 cgccccctat tgacgtcaat gacggtaaat ggcccgcctg gcattgtgcc cagtacatga
     1801 ccttatggga ctttcctact tggcagtaca tctacgtatt agtcatcgct attaccatgg
     1861 tcgaggtgag ccccacgttc tgcttcactc tccccatctc ccccccctcc ccacccccaa
     1921 ttttgtattt atttattttt taattatttt gtgcagcgat gggggcgggg gggggggggg
     1981 ggcgcgcgcc rggsggggsg ggggggggsg gggggsgggg sggggsgrgg sggrgaggkg
     2041 cggcggcagc caatcagagc ggcgcgctcc gaaagtttcc ttttatggcg aggcggcggc
     2101 ggcggcggcc ctataaaaag cgaagcgcgc ggcgggcggg agtcgctgcg cgctgccttc
     2161 gccccgtgcc ccgctccgcc gccgcctcgc gccgcccgcc ccggctctga ctgaccgcgt
     2221 tactcccaca ggtgagcggg cgggacggcc cttctcctcc gggctgtaat tagctgagca
     2281 agaggtaagg gtttaaggga tggttggttg gtggggtatt aatgtttaat tacctggagc
     2341 acctgcctga aatcactttt tttcaggttg gaccggtgcc accatggact ataaggacca
     2401 cgacggagac tacaaggatc atgatattga ttacaaagac gatgacgata agatggcccc
     2461 aaagaagaag cggaaggtcg gtatccacgg agtcccagca gccgacaaga agtacagcat
     2521 cggcctggac atcggcacca actctgtggg ctgggccgtg atcaccgacg agtacaaggt
     2581 gcccagcaag aaattcaagg tgctgggcaa caccgaccgg cacagcatca agaagaacct
     2641 gatcggagcc ctgctgttcg acagcggcga aacagccgag gccacccggc tgaagagaac
     2701 cgccagaaga agatacacca gacggaagaa ccggatctgc tatctgcaag agatcttcag
     2761 caacgagatg gccaaggtgg acgacagctt cttccacaga ctggaagagt ccttcctggt
     2821 ggaagaggat aagaagcacg agcggcaccc catcttcggc aacatcgtgg acgaggtggc
     2881 ctaccacgag aagtacccca ccatctacca cctgagaaag aaactggtgg acagcaccga
     2941 caaggccgac ctgcggctga tctatctggc cctggcccac atgatcaagt tccggggcca
     3001 cttcctgatc gagggcgacc tgaaccccga caacagcgac gtggacaagc tgttcatcca
     3061 gctggtgcag acctacaacc agctgttcga ggaaaacccc atcaacgcca gcggcgtgga
     3121 cgccaaggcc atcctgtctg ccagactgag caagagcaga cggctggaaa atctgatcgc
     3181 ccagctgccc ggcgagaaga agaatggcct gttcggaaac ctgattgccc tgagcctggg
     3241 cctgaccccc aacttcaaga gcaacttcga cctggccgag gatgccaaac tgcagctgag
     3301 caaggacacc tacgacgacg acctggacaa cctgctggcc cagatcggcg accagtacgc
     3361 cgacctgttt ctggccgcca agaacctgtc cgacgccatc ctgctgagcg acatcctgag
     3421 agtgaacacc gagatcacca aggcccccct gagcgcctct atgatcaaga gatacgacga
     3481 gcaccaccag gacctgaccc tgctgaaagc tctcgtgcgg cagcagctgc ctgagaagta
     3541 caaagagatt ttcttcgacc agagcaagaa cggctacgcc ggctacattg acggcggagc
     3601 cagccaggaa gagttctaca agttcatcaa gcccatcctg gaaaagatgg acggcaccga
     3661 ggaactgctc gtgaagctga acagagagga cctgctgcgg aagcagcgga ccttcgacaa
     3721 cggcagcatc ccccaccaga tccacctggg agagctgcac gccattctgc ggcggcagga
     3781 agatttttac ccattcctga aggacaaccg ggaaaagatc gagaagatcc tgaccttccg
     3841 catcccctac tacgtgggcc ctctggccag gggaaacagc agattcgcct ggatgaccag
     3901 aaagagcgag gaaaccatca ccccctggaa cttcgaggaa gtggtggaca agggcgcttc
     3961 cgcccagagc ttcatcgagc ggatgaccaa cttcgataag aacctgccca acgagaaggt
     4021 gctgcccaag cacagcctgc tgtacgagta cttcaccgtg tataacgagc tgaccaaagt
     4081 gaaatacgtg accgagggaa tgagaaagcc cgccttcctg agcggcgagc agaaaaaggc
     4141 catcgtggac ctgctgttca agaccaaccg gaaagtgacc gtgaagcagc tgaaagagga
     4201 ctacttcaag aaaatcgagt gcttcgactc cgtggaaatc tccggcgtgg aagatcggtt
     4261 caacgcctcc ctgggcacat accacgatct gctgaaaatt atcaaggaca aggacttcct
     4321 ggacaatgag gaaaacgagg acattctgga agatatcgtg ctgaccctga cactgtttga
     4381 ggacagagag atgatcgagg aacggctgaa aacctatgcc cacctgttcg acgacaaagt
     4441 gatgaagcag ctgaagcggc ggagatacac cggctggggc aggctgagcc ggaagctgat
     4501 caacggcatc cgggacaagc agtccggcaa gacaatcctg gatttcctga agtccgacgg
     4561 cttcgccaac agaaacttca tgcagctgat ccacgacgac agcctgacct ttaaagagga
     4621 catccagaaa gcccaggtgt ccggccaggg cgatagcctg cacgagcaca ttgccaatct
     4681 ggccggcagc cccgccatta agaagggcat cctgcagaca gtgaaggtgg tggacgagct
     4741 cgtgaaagtg atgggccggc acaagcccga gaacatcgtg atcgaaatgg ccagagagaa
     4801 ccagaccacc cagaagggac agaagaacag ccgcgagaga atgaagcgga tcgaagaggg
     4861 catcaaagag ctgggcagcc agatcctgaa agaacacccc gtggaaaaca cccagctgca
     4921 gaacgagaag ctgtacctgt actacctgca gaatgggcgg gatatgtacg tggaccagga
     4981 actggacatc aaccggctgt ccgactacga tgtggaccat atcgtgcctc agagctttct
     5041 gaaggacgac tccatcgaca acaaggtgct gaccagaagc gacaagaacc ggggcaagag
     5101 cgacaacgtg ccctccgaag aggtcgtgaa gaagatgaag aactactggc ggcagctgct
     5161 gaacgccaag ctgattaccc agagaaagtt cgacaatctg accaaggccg agagaggcgg
     5221 cctgagcgaa ctggataagg ccggcttcat caagagacag ctggtggaaa cccggcagat
     5281 cacaaagcac gtggcacaga tcctggactc ccggatgaac actaagtacg acgagaatga
     5341 caagctgatc cgggaagtga aagtgatcac cctgaagtcc aagctggtgt ccgatttccg
     5401 gaaggatttc cagttttaca aagtgcgcga gatcaacaac taccaccacg cccacgacgc
     5461 ctacctgaac gccgtcgtgg gaaccgccct gatcaaaaag taccctaagc tggaaagcga
     5521 gttcgtgtac ggcgactaca aggtgtacga cgtgcggaag atgatcgcca agagcgagca
     5581 ggaaatcggc aaggctaccg ccaagtactt cttctacagc aacatcatga actttttcaa
     5641 gaccgagatt accctggcca acggcgagat ccggaagcgg cctctgatcg agacaaacgg
     5701 cgaaaccggg gagatcgtgt gggataaggg ccgggatttt gccaccgtgc ggaaagtgct
     5761 gagcatgccc caagtgaata tcgtgaaaaa gaccgaggtg cagacaggcg gcttcagcaa
     5821 agagtctatc ctgcccaaga ggaacagcga taagctgatc gccagaaaga aggactggga
     5881 ccctaagaag tacggcggct tcgacagccc caccgtggcc tattctgtgc tggtggtggc
     5941 caaagtggaa aagggcaagt ccaagaaact gaagagtgtg aaagagctgc tggggatcac
     6001 catcatggaa agaagcagct tcgagaagaa tcccatcgac tttctggaag ccaagggcta
     6061 caaagaagtg aaaaaggacc tgatcatcaa gctgcctaag tactccctgt tcgagctgga
     6121 aaacggccgg aagagaatgc tggcctctgc cggcgaactg cagaagggaa acgaactggc
     6181 cctgccctcc aaatatgtga acttcctgta cctggccagc cactatgaga agctgaaggg
     6241 ctcccccgag gataatgagc agaaacagct gtttgtggaa cagcacaagc actacctgga
     6301 cgagatcatc gagcagatca gcgagttctc caagagagtg atcctggccg acgctaatct
     6361 ggacaaagtg ctgtccgcct acaacaagca ccgggataag cccatcagag agcaggccga
     6421 gaatatcatc cacctgttta ccctgaccaa tctgggagcc cctgccgcct tcaagtactt
     6481 tgacaccacc atcgaccgga agaggtacac cagcaccaaa gaggtgctgg acgccaccct
     6541 gatccaccag agcatcaccg gcctgtacga gacacggatc gacctgtctc agctgggagg
     6601 cgacaaaagg ccggcggcca cgaaaaaggc cggccaggca aaaaagaaaa agtaagaatt
     6661 cctagagctc gctgatcagc ctcgactgtg ccttctagtt gccagccatc tgttgtttgc
     6721 ccctcccccg tgccttcctt gaccctggaa ggtgccactc ccactgtcct ttcctaataa
     6781 aatgaggaaa ttgcatcgca ttgtctgagt aggtgtcatt ctattctggg gggtggggtg
     6841 gggcaggaca gcaaggggga ggattgggaa gagaatagca ggcatgctgg ggagcggccg
     6901 caggaacccc tagtgatgga gttggccact ccctctctgc gcgctcgctc gctcactgag
     6961 gccgggcgac caaaggtcgc ccgacgcccg ggctttgccc gggcggcctc agtgagcgag
     7021 cgagcgcgca gctgcctgca ggggcgcctg atgcggtatt ttctccttac gcatctgtgc
     7081 ggtatttcac accgcatacg tcaaagcaac catagtacgc gccctgtagc ggcgcattaa
     7141 gcgcggcggg tgtggtggtt acgcgcagcg tgaccgctac acttgccagc gccttagcgc
     7201 ccgctccttt cgctttcttc ccttcctttc tcgccacgtt cgccggcttt ccccgtcaag
     7261 ctctaaatcg ggggctccct ttagggttcc gatttagtgc tttacggcac ctcgacccca
     7321 aaaaacttga tttgggtgat ggttcacgta gtgggccatc gccctgatag acggtttttc
     7381 gccctttgac gttggagtcc acgttcttta atagtggact cttgttccaa actggaacaa
     7441 cactcaactc tatctcgggc tattcttttg atttataagg gattttgccg atttcggtct
     7501 attggttaaa aaatgagctg atttaacaaa aatttaacgc gaattttaac aaaatattaa
     7561 cgtttacaat tttatggtgc actctcagta caatctgctc tgatgccgca tagttaagcc
     7621 agccccgaca cccgccaaca cccgctgacg cgccctgacg ggcttgtctg ctcccggcat
     7681 ccgcttacag acaagctgtg accgtctccg ggagctgcat gtgtcagagg ttttcaccgt
     7741 catcaccgaa acgcgcgaga cgaaagggcc tcgtgatacg cctattttta taggttaatg
     7801 tcatgataat aatggtttct tagacgtcag gtggcacttt tcggggaaat gtgcgcggaa
     7861 cccctatttg tttatttttc taaatacatt caaatatgta tccgctcatg agacaataac
     7921 cctgataaat gcttcaataa tattgaaaaa ggaagagtat gagtattcaa catttccgtg
     7981 tcgcccttat tccctttttt gcggcatttt gccttcctgt ttttgctcac ccagaaacgc
     8041 tggtgaaagt aaaagatgct gaagatcagt tgggtgcacg agtgggttac atcgaactgg
     8101 atctcaacag cggtaagatc cttgagagtt ttcgccccga agaacgtttt ccaatgatga
     8161 gcacttttaa agttctgcta tgtggcgcgg tattatcccg tattgacgcc gggcaagagc
     8221 aactcggtcg ccgcatacac tattctcaga atgacttggt tgagtactca ccagtcacag
     8281 aaaagcatct tacggatggc atgacagtaa gagaattatg cagtgctgcc ataaccatga
     8341 gtgataacac tgcggccaac ttacttctga caacgatcgg aggaccgaag gagctaaccg
     8401 cttttttgca caacatgggg gatcatgtaa ctcgccttga tcgttgggaa ccggagctga
     8461 atgaagccat accaaacgac gagcgtgaca ccacgatgcc tgtagcaatg gcaacaacgt
     8521 tgcgcaaact attaactggc gaactactta ctctagcttc ccggcaacaa ttaatagact
     8581 ggatggaggc ggataaagtt gcaggaccac ttctgcgctc ggcccttccg gctggctggt
     8641 ttattgctga taaatctgga gccggtgagc gtggaagccg cggtatcatt gcagcactgg
     8701 ggccagatgg taagccctcc cgtatcgtag ttatctacac gacggggagt caggcaacta
     8761 tggatgaacg aaatagacag atcgctgaga taggtgcctc actgattaag cattggtaac
     8821 tgtcagacca agtttactca tatatacttt agattgattt aaaacttcat ttttaattta
     8881 aaaggatcta ggtgaagatc ctttttgata atctcatgac caaaatccct taacgtgagt
     8941 tttcgttcca
//
