LOCUS       sequence-447743-        9940 bp    DNA     circular SYN 15-DEC-2025
DEFINITION  It contains a ccdB-CMr negative selection module flanked by BpiI 
            (BbsI) sites that allows excluding clones without correct insertion 
            of sgRNA. Human codon optimized Cas9 expressing plasmid..
ACCESSION   .
VERSION     .
KEYWORDS    .
SOURCE      synthetic DNA construct
  ORGANISM  synthetic DNA construct
REFERENCE   1  (bases 1 to 9940)
  AUTHORS   Petruskeviciute A, Simuliunaite U, Polanco CM, Rojas B, Kuras S, 
            Valatkaite-Rakstiene B, Norvilas R, Vijaya AK, Munoz P, Neniskyte U,
            Jakubauskas A, Burokas A, Nalvarte I, Inzunza J, Naumovas D, Stoskus
            M, Griskevicius L, Baltriukiene D, Arias J
  TITLE     Generation of a genetically encoded voltage indicator MARINA 
            reporter human iPS cell line using Cas9 (VULSCi002-A-2).
  JOURNAL   Stem Cell Res. 2024 Dec 12;82:103628. doi: 
            10.1016/j.scr.2024.103628.
  PUBMED    39721357
REFERENCE   2  (bases 1 to 9940)
  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: "Stem Cell 
            Res."; date: "2024-12-12"; volume: "82:103628. doi"; pages: " 
            10.1016/j.scr.2024.103628"
COMMENT     Sequence Label: pX330-U6-(negative selection
            module)-Chimeric_...
FEATURES             Location/Qualifiers
     source          1..9940
                     /mol_type="other DNA"
                     /organism="synthetic DNA construct"
     promoter        1..249
                     /label=U6 promoter
                     /note="RNA polymerase III promoter for human U6 snRNA 
                     (Domitrovich & Kunkel, 2003)"
     primer_bind     1..21
                     /label=hU6-F
                     /note="Human U6 promoter, forward primer"
     primer_bind     172..191
                     /label=LKO.1 5'
                     /note="Human U6 promoter, forward primer"
     promoter        join(267..272,273..290,291..297)
                     /label=lac UV5 promoter
                     /note="E. coli lac promoter with an ""up"" mutation"
     CDS             351..1031
                     /codon_start=1
                     /product="chloramphenicol acetyltransferase"
                     /label=CmR
                     /note="confers resistance to chloramphenicol"
                     /translation="MEKKITGYTTVDISQWHRKEHFEAFQSVAQCTYNQTVQLDITAFL
                     KTVKKNKHKFYPAFIHILARLMNAHPEFRMAMKDGELVIWDSVHPCYTVFHEQTETFSS
                     LWSEYHDDFRQFLHIYSQDVACYGENLAYFPKGFIENMFFVSANPWVSFTSFDLNVANM
                     DNFFAPVFTMGKYYTQGDKVLMPLAIQVHHAVCDGFHVGRMLNELQQYCDEWQAGRNLE
                     DPAY"
     primer_bind     complement(417..436)
                     /label=CAT-R
                     /note="Chloramphenicol resistance gene, reverse primer"
     CDS             1351..1656
                     /codon_start=1
                     /gene="ccdB"
                     /product="CcdB, a bacterial toxin that poisons DNA gyrase"
                     /label=ccdB
                     /note="Plasmids containing the ccdB gene cannot be 
                     propagated in standard E. coli strains."
                     /translation="MQFKVYTYKRESRYRLFVDVQSDIIDTPGRRMVIPLASARLLSDK
                     VPRELYPVVHIGDESWRMMTTDMASVPVSVIGEEVADLSHRENDIKNAINLMFWGI"
     primer_bind     1535..1554
                     /label=ccdB-fwd
                     /note="ccdB gene, forward primer"
     misc_RNA        1704..1779
                     /label=gRNA scaffold
                     /note="guide RNA scaffold for the Streptococcus pyogenes 
                     CRISPR/Cas9 system"
     enhancer        1876..2161
                     /label=CMV enhancer
                     /note="human cytomegalovirus immediate early enhancer; 
                     contains an 18-bp deletion relative to the standard CMV 
                     enhancer"
     promoter        2163..2439
                     /label=chicken beta-actin promoter
     intron          2440..2667
                     /label=hybrid intron
                     /note="hybrid between chicken beta-actin (CBA) and minute 
                     virus of mice (MMV) introns (Gray et al., 2011)"
     regulatory      2679..2688
                     /label=Kozak sequence
                     /note="vertebrate consensus sequence for strong initiation 
                     of translation (Kozak, 1987)"
                     /regulatory_class="other"
     CDS             2688..2753
                     /codon_start=1
                     /product="three tandem FLAG(R) epitope tags, followed by an
                     enterokinase cleavage site"
                     /label=3xFLAG
                     /translation="DYKDHDGDYKDHDIDYKDDDDK"
     CDS             2760..2780
                     /codon_start=1
                     /product="nuclear localization signal of SV40 (simian virus
                     40) large T antigen"
                     /label=SV40 NLS
                     /translation="PKKKRKV"
     CDS             2805..6905
                     /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             2805..6905
                     /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             6906..6953
                     /codon_start=1
                     /product="bipartite nuclear localization signal from 
                     nucleoplasmin"
                     /label=nucleoplasmin NLS
                     /translation="KRPAATKKAGQAKKKK"
     primer_bind     complement(6981..6998)
                     /label=BGH-rev
                     /note="Bovine growth hormone terminator, reverse primer. 
                     Also called BGH reverse"
     polyA_signal    6987..7194
                     /label=bGH poly(A) signal
                     /note="bovine growth hormone polyadenylation signal"
     repeat_region   7203..7343
                     /label=AAV2 ITR
                     /note="inverted terminal repeat of adeno-associated virus 
                     serotype 2"
     repeat_region   7203..7332
                     /label=AAV2 ITR
     rep_origin      7418..7873
                     /direction=RIGHT
                     /label=f1 ori
                     /note="f1 bacteriophage origin of replication; arrow 
                     indicates direction of (+) strand synthesis"
     primer_bind     complement(7505..7524)
                     /label=F1ori-R
                     /note="F1 origin, reverse primer"
     primer_bind     7715..7736
                     /label=F1ori-F
                     /note="F1 origin, forward primer"
     primer_bind     complement(7890..7909)
                     /label=pRS-marker
                     /note="pRS vectors, use to sequence yeast selectable 
                     marker"
     primer_bind     8009..8031
                     /label=pGEX 3'
                     /note="pGEX vectors, reverse primer"
     primer_bind     complement(8069..8087)
                     /label=pBRforEco
                     /note="pBR322 vectors, upsteam of EcoRI site, forward 
                     primer"
     promoter        8155..8259
                     /gene="bla"
                     /label=AmpR promoter
     CDS             join(8260..8328,8329..9120)
                     /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(8478..8497)
                     /label=Amp-R
                     /note="Ampicillin resistance gene, reverse primer"
     rep_origin      9291..9879
                     /direction=RIGHT
                     /label=ori
                     /note="high-copy-number ColE1/pMB1/pBR322/pUC origin of 
                     replication"
     primer_bind     9780..9799
                     /label=pBR322ori-F
                     /note="pBR322 origin, forward primer"
ORIGIN
        1 gagggcctat ttcccatgat tccttcatat ttgcatatac gatacaaggc tgttagagag
       61 ataattggaa ttaatttgac tgtaaacaca aagatattag tacaaaatac gtgacgtaga
      121 aagtaataat ttcttgggta gtttgcagtt ttaaaattat gttttaaaat ggactatcat
      181 atgcttaccg taacttgaaa gtatttcgat ttcttggctt tatatatctt gtggaaagga
      241 cgaaacaccg ggtcttcgcc ccaggcttta cactttatgc ttccggctcg tataatgtgt
      301 ggattttgag ttaggatccg tcgagatttt caggagctaa ggaagctaaa atggagaaaa
      361 aaatcactgg atataccacc gttgatatat cccaatggca tcgtaaagaa cattttgagg
      421 catttcagtc agttgctcaa tgtacctata accagaccgt tcagctggat attacggcct
      481 ttttaaagac cgtaaagaaa aataagcaca agttttatcc ggcctttatt cacattcttg
      541 cccgcctgat gaatgctcat ccggaattcc gtatggcaat gaaagacggt gagctggtga
      601 tatgggatag tgttcaccct tgttacaccg ttttccatga gcaaactgaa acgttttcat
      661 cgctctggag tgaataccac gacgatttcc ggcagtttct acacatatat tcgcaagatg
      721 tggcgtgtta cggtgaaaac ctggcctatt tccctaaagg gtttattgag aatatgtttt
      781 tcgtctcagc caatccctgg gtgagtttca ccagttttga tttaaacgtg gccaatatgg
      841 acaacttctt cgcccccgtt ttcaccatgg gcaaatatta tacgcaaggc gacaaggtgc
      901 tgatgccgct ggcgattcag gttcatcatg ccgtttgtga tggcttccat gtcggcagaa
      961 tgcttaatga attacaacag tactgcgatg agtggcaggc ggggcgtaat ctagaggatc
     1021 cggcttacta aaagccagat aacagtatgc gtatttgcgc gctgattttt gcggtataag
     1081 aatatatact gatatgtata cccgaagtat gtcaaaaaga ggtatgctat gaagcagcgt
     1141 attacagtga cagttgacag cgacagctat cagttgctca aggcatatat gatgtcaata
     1201 tctccggtct ggtaagcaca accatgcaga atgaagcccg tcgtctgcgt gccgaacgct
     1261 ggaaagcgga aaatcaggaa gggatggctg aggtcgcccg gtttattgaa atgaacggct
     1321 cttttgctga cgagaacagg ggctggtgaa atgcagttta aggtttacac ctataaaaga
     1381 gagagccgtt atcgtctgtt tgtggatgta cagagtgata ttattgacac gcccgggcga
     1441 cggatggtga tccccctggc cagtgcacgt ctgctgtcag ataaagtccc ccgtgaactt
     1501 tacccggtgg tgcatatcgg ggatgaaagc tggcgcatga tgaccaccga tatggccagt
     1561 gtgccggtct ccgttatcgg ggaagaagtg gctgatctca gccaccgcga aaatgacatc
     1621 aaaaacgcca ttaacctgat gttctgggga atataaatgt caggctccct tatacacagc
     1681 cagtctgcag gtcgagaaga cctgttttag agctagaaat agcaagttaa aataaggcta
     1741 gtccgttatc aacttgaaaa agtggcaccg agtcggtgct tttttgtttt agagctagaa
     1801 atagcaagtt aaaataaggc tagtccgttt ttagcgcgtg cgccaattct gcagacaaat
     1861 ggctctagag gtacccgtta cataacttac ggtaaatggc ccgcctggct gaccgcccaa
     1921 cgacccccgc ccattgacgt caatagtaac gccaataggg actttccatt gacgtcaatg
     1981 ggtggagtat ttacggtaaa ctgcccactt ggcagtacat caagtgtatc atatgccaag
     2041 tacgccccct attgacgtca atgacggtaa atggcccgcc tggcattgtg cccagtacat
     2101 gaccttatgg gactttccta cttggcagta catctacgta ttagtcatcg ctattaccat
     2161 ggtcgaggtg agccccacgt tctgcttcac tctccccatc tcccccccct ccccaccccc
     2221 aattttgtat ttatttattt tttaattatt ttgtgcagcg atgggggcgg gggggggggg
     2281 gggsgcgcgc cagggggggg gggggggggs gaggggsggg gsggggsgag gsggagaggt
     2341 gcggcggcag ccaatcagag cggcgcgctc cgaaagtttc cttttatggc gaggcggcgg
     2401 cggcggcggc cctataaaaa gcgaagcgcg cggcgggcgg gagtcgctgc gcgctgcctt
     2461 cgccccgtgc cccgctccgc cgccgcctcg cgccgcccgc cccggctctg actgaccgcg
     2521 ttactcccac aggtgagcgg gcgggacggc ccttctcctc cgggctgtaa ttagctgagc
     2581 aagaggtaag ggtttaaggg atggttggtt ggtggggtat taatgtttaa ttacctggag
     2641 cacctgcctg aaatcacttt ttttcaggtt ggaccggtgc caccatggac tataaggacc
     2701 acgacggaga ctacaaggat catgatattg attacaaaga cgatgacgat aagatggccc
     2761 caaagaagaa gcggaaggtc ggtatccacg gagtcccagc agccgacaag aagtacagca
     2821 tcggcctgga catcggcacc aactctgtgg gctgggccgt gatcaccgac gagtacaagg
     2881 tgcccagcaa gaaattcaag gtgctgggca acaccgaccg gcacagcatc aagaagaacc
     2941 tgatcggagc cctgctgttc gacagcggcg aaacagccga ggccacccgg ctgaagagaa
     3001 ccgccagaag aagatacacc agacggaaga accggatctg ctatctgcaa gagatcttca
     3061 gcaacgagat ggccaaggtg gacgacagct tcttccacag actggaagag tccttcctgg
     3121 tggaagagga taagaagcac gagcggcacc ccatcttcgg caacatcgtg gacgaggtgg
     3181 cctaccacga gaagtacccc accatctacc acctgagaaa gaaactggtg gacagcaccg
     3241 acaaggccga cctgcggctg atctatctgg ccctggccca catgatcaag ttccggggcc
     3301 acttcctgat cgagggcgac ctgaaccccg acaacagcga cgtggacaag ctgttcatcc
     3361 agctggtgca gacctacaac cagctgttcg aggaaaaccc catcaacgcc agcggcgtgg
     3421 acgccaaggc catcctgtct gccagactga gcaagagcag acggctggaa aatctgatcg
     3481 cccagctgcc cggcgagaag aagaatggcc tgttcggaaa cctgattgcc ctgagcctgg
     3541 gcctgacccc caacttcaag agcaacttcg acctggccga ggatgccaaa ctgcagctga
     3601 gcaaggacac ctacgacgac gacctggaca acctgctggc ccagatcggc gaccagtacg
     3661 ccgacctgtt tctggccgcc aagaacctgt ccgacgccat cctgctgagc gacatcctga
     3721 gagtgaacac cgagatcacc aaggcccccc tgagcgcctc tatgatcaag agatacgacg
     3781 agcaccacca ggacctgacc ctgctgaaag ctctcgtgcg gcagcagctg cctgagaagt
     3841 acaaagagat tttcttcgac cagagcaaga acggctacgc cggctacatt gacggcggag
     3901 ccagccagga agagttctac aagttcatca agcccatcct ggaaaagatg gacggcaccg
     3961 aggaactgct cgtgaagctg aacagagagg acctgctgcg gaagcagcgg accttcgaca
     4021 acggcagcat cccccaccag atccacctgg gagagctgca cgccattctg cggcggcagg
     4081 aagattttta cccattcctg aaggacaacc gggaaaagat cgagaagatc ctgaccttcc
     4141 gcatccccta ctacgtgggc cctctggcca ggggaaacag cagattcgcc tggatgacca
     4201 gaaagagcga ggaaaccatc accccctgga acttcgagga agtggtggac aagggcgctt
     4261 ccgcccagag cttcatcgag cggatgacca acttcgataa gaacctgccc aacgagaagg
     4321 tgctgcccaa gcacagcctg ctgtacgagt acttcaccgt gtataacgag ctgaccaaag
     4381 tgaaatacgt gaccgaggga atgagaaagc ccgccttcct gagcggcgag cagaaaaagg
     4441 ccatcgtgga cctgctgttc aagaccaacc ggaaagtgac cgtgaagcag ctgaaagagg
     4501 actacttcaa gaaaatcgag tgcttcgact ccgtggaaat ctccggcgtg gaagatcggt
     4561 tcaacgcctc cctgggcaca taccacgatc tgctgaaaat tatcaaggac aaggacttcc
     4621 tggacaatga ggaaaacgag gacattctgg aagatatcgt gctgaccctg acactgtttg
     4681 aggacagaga gatgatcgag gaacggctga aaacctatgc ccacctgttc gacgacaaag
     4741 tgatgaagca gctgaagcgg cggagataca ccggctgggg caggctgagc cggaagctga
     4801 tcaacggcat ccgggacaag cagtccggca agacaatcct ggatttcctg aagtccgacg
     4861 gcttcgccaa cagaaacttc atgcagctga tccacgacga cagcctgacc tttaaagagg
     4921 acatccagaa agcccaggtg tccggccagg gcgatagcct gcacgagcac attgccaatc
     4981 tggccggcag ccccgccatt aagaagggca tcctgcagac agtgaaggtg gtggacgagc
     5041 tcgtgaaagt gatgggccgg cacaagcccg agaacatcgt gatcgaaatg gccagagaga
     5101 accagaccac ccagaaggga cagaagaaca gccgcgagag aatgaagcgg atcgaagagg
     5161 gcatcaaaga gctgggcagc cagatcctga aagaacaccc cgtggaaaac acccagctgc
     5221 agaacgagaa gctgtacctg tactacctgc agaatgggcg ggatatgtac gtggaccagg
     5281 aactggacat caaccggctg tccgactacg atgtggacca tatcgtgcct cagagctttc
     5341 tgaaggacga ctccatcgac aacaaggtgc tgaccagaag cgacaagaac cggggcaaga
     5401 gcgacaacgt gccctccgaa gaggtcgtga agaagatgaa gaactactgg cggcagctgc
     5461 tgaacgccaa gctgattacc cagagaaagt tcgacaatct gaccaaggcc gagagaggcg
     5521 gcctgagcga actggataag gccggcttca tcaagagaca gctggtggaa acccggcaga
     5581 tcacaaagca cgtggcacag atcctggact cccggatgaa cactaagtac gacgagaatg
     5641 acaagctgat ccgggaagtg aaagtgatca ccctgaagtc caagctggtg tccgatttcc
     5701 ggaaggattt ccagttttac aaagtgcgcg agatcaacaa ctaccaccac gcccacgacg
     5761 cctacctgaa cgccgtcgtg ggaaccgccc tgatcaaaaa gtaccctaag ctggaaagcg
     5821 agttcgtgta cggcgactac aaggtgtacg acgtgcggaa gatgatcgcc aagagcgagc
     5881 aggaaatcgg caaggctacc gccaagtact tcttctacag caacatcatg aactttttca
     5941 agaccgagat taccctggcc aacggcgaga tccggaagcg gcctctgatc gagacaaacg
     6001 gcgaaaccgg ggagatcgtg tgggataagg gccgggattt tgccaccgtg cggaaagtgc
     6061 tgagcatgcc ccaagtgaat atcgtgaaaa agaccgaggt gcagacaggc ggcttcagca
     6121 aagagtctat cctgcccaag aggaacagcg ataagctgat cgccagaaag aaggactggg
     6181 accctaagaa gtacggcggc ttcgacagcc ccaccgtggc ctattctgtg ctggtggtgg
     6241 ccaaagtgga aaagggcaag tccaagaaac tgaagagtgt gaaagagctg ctggggatca
     6301 ccatcatgga aagaagcagc ttcgagaaga atcccatcga ctttctggaa gccaagggct
     6361 acaaagaagt gaaaaaggac ctgatcatca agctgcctaa gtactccctg ttcgagctgg
     6421 aaaacggccg gaagagaatg ctggcctctg ccggcgaact gcagaaggga aacgaactgg
     6481 ccctgccctc caaatatgtg aacttcctgt acctggccag ccactatgag aagctgaagg
     6541 gctcccccga ggataatgag cagaaacagc tgtttgtgga acagcacaag cactacctgg
     6601 acgagatcat cgagcagatc agcgagttct ccaagagagt gatcctggcc gacgctaatc
     6661 tggacaaagt gctgtccgcc tacaacaagc accgggataa gcccatcaga gagcaggccg
     6721 agaatatcat ccacctgttt accctgacca atctgggagc ccctgccgcc ttcaagtact
     6781 ttgacaccac catcgaccgg aagaggtaca ccagcaccaa agaggtgctg gacgccaccc
     6841 tgatccacca gagcatcacc ggcctgtacg agacacggat cgacctgtct cagctgggag
     6901 gcgacaaaag gccggcggcc acgaaaaagg ccggccaggc aaaaaagaaa aagtaagaat
     6961 tcctagagct cgctgatcag cctcgactgt gccttctagt tgccagccat ctgttgtttg
     7021 cccctccccc gtgccttcct tgaccctgga aggtgccact cccactgtcc tttcctaata
     7081 aaatgaggaa attgcatcgc attgtctgag taggtgtcat tctattctgg ggggtggggt
     7141 ggggcaggac agcaaggggg aggattggga agagaatagc aggcatgctg gggagcggcc
     7201 gcaggaaccc ctagtgatgg agttggccac tccctctctg cgcgctcgct cgctcactga
     7261 ggccgggcga ccaaaggtcg cccgacgccc gggctttgcc cgggcggcct cagtgagcga
     7321 gcgagcgcgc agctgcctgc aggggcgcct gatgcggtat tttctcctta cgcatctgtg
     7381 cggtatttca caccgcatac gtcaaagcaa ccatagtacg cgccctgtag cggcgcatta
     7441 agcgcggcgg gtgtggtggt tacgcgcagc gtgaccgcta cacttgccag cgccttagcg
     7501 cccgctcctt tcgctttctt cccttccttt ctcgccacgt tcgccggctt tccccgtcaa
     7561 gctctaaatc gggggctccc tttagggttc cgatttagtg ctttacggca cctcgacccc
     7621 aaaaaacttg atttgggtga tggttcacgt agtgggccat cgccctgata gacggttttt
     7681 cgccctttga cgttggagtc cacgttcttt aatagtggac tcttgttcca aactggaaca
     7741 acactcaact ctatctcggg ctattctttt gatttataag ggattttgcc gatttcggtc
     7801 tattggttaa aaaatgagct gatttaacaa aaatttaacg cgaattttaa caaaatatta
     7861 acgtttacaa ttttatggtg cactctcagt acaatctgct ctgatgccgc atagttaagc
     7921 cagccccgac acccgccaac acccgctgac gcgccctgac gggcttgtct gctcccggca
     7981 tccgcttaca gacaagctgt gaccgtctcc gggagctgca tgtgtcagag gttttcaccg
     8041 tcatcaccga aacgcgcgag acgaaagggc ctcgtgatac gcctattttt ataggttaat
     8101 gtcatgataa taatggtttc ttagacgtca ggtggcactt ttcggggaaa tgtgcgcgga
     8161 acccctattt gtttattttt ctaaatacat tcaaatatgt atccgctcat gagacaataa
     8221 ccctgataaa tgcttcaata atattgaaaa aggaagagta tgagtattca acatttccgt
     8281 gtcgccctta ttcccttttt tgcggcattt tgccttcctg tttttgctca cccagaaacg
     8341 ctggtgaaag taaaagatgc tgaagatcag ttgggtgcac gagtgggtta catcgaactg
     8401 gatctcaaca gcggtaagat ccttgagagt tttcgccccg aagaacgttt tccaatgatg
     8461 agcactttta aagttctgct atgtggcgcg gtattatccc gtattgacgc cgggcaagag
     8521 caactcggtc gccgcataca ctattctcag aatgacttgg ttgagtactc accagtcaca
     8581 gaaaagcatc ttacggatgg catgacagta agagaattat gcagtgctgc cataaccatg
     8641 agtgataaca ctgcggccaa cttacttctg acaacgatcg gaggaccgaa ggagctaacc
     8701 gcttttttgc acaacatggg ggatcatgta actcgccttg atcgttggga accggagctg
     8761 aatgaagcca taccaaacga cgagcgtgac accacgatgc ctgtagcaat ggcaacaacg
     8821 ttgcgcaaac tattaactgg cgaactactt actctagctt cccggcaaca attaatagac
     8881 tggatggagg cggataaagt tgcaggacca cttctgcgct cggcccttcc ggctggctgg
     8941 tttattgctg ataaatctgg agccggtgag cgtggaagcc gcggtatcat tgcagcactg
     9001 gggccagatg gtaagccctc ccgtatcgta gttatctaca cgacggggag tcaggcaact
     9061 atggatgaac gaaatagaca gatcgctgag ataggtgcct cactgattaa gcattggtaa
     9121 ctgtcagacc aagtttactc atatatactt tagattgatt taaaacttca tttttaattt
     9181 aaaaggatct aggtgaagat cctttttgat aatctcatga ccaaaatccc ttaacgtgag
     9241 ttttcgttcc actgagcgtc agaccccgta gaaaagatca aaggatcttc ttgagatcct
     9301 ttttttctgc gcgtaatctg ctgcttgcaa acaaaaaaac caccgctacc agcggtggtt
     9361 tgtttgccgg atcaagagct accaactctt tttccgaagg taactggctt cagcagagcg
     9421 cagataccaa atactgttct tctagtgtag ccgtagttag gccaccactt caagaactct
     9481 gtagcaccgc ctacatacct cgctctgcta atcctgttac cagtggctgc tgccagtggc
     9541 gataagtcgt gtcttaccgg gttggactca agacgatagt taccggataa ggcgcagcgg
     9601 tcgggctgaa cggggggttc gtgcacacag cccagcttgg agcgaacgac ctacaccgaa
     9661 ctgagatacc tacagcgtga gctatgagaa agcgccacgc ttcccgaagg gagaaaggcg
     9721 gacaggtatc cggtaagcgg cagggtcgga acaggagagc gcacgaggga gcttccaggg
     9781 ggaaacgcct ggtatcttta tagtcctgtc gggtttcgcc acctctgact tgagcgtcga
     9841 tttttgtgat gctcgtcagg ggggcggagc ctatggaaaa acgccagcaa cgcggccttt
     9901 ttacggttcc tggccttttg ctggcctttt gctcacatgt
//
