LOCUS       sequence-328047-        9305 bp    DNA     circular SYN 15-DEC-2025
DEFINITION  Expresses sgRNA targeting human GJA1 locus.
ACCESSION   .
VERSION     .
KEYWORDS    .
SOURCE      synthetic DNA construct
  ORGANISM  synthetic DNA construct
REFERENCE   1  (bases 1 to 9305)
  AUTHORS   Cheng L, Cring MR, Wadkins DA, Kuehn MH
  TITLE     Absence of Connexin 43 results in smaller retinas and arrested, 
            depolarized retinal progenitor cells in human retinal organoids.
  JOURNAL   Stem Cells. 2022 Mar 9. pii: 6545973. doi: 10.1093/stmcls/sxac017.
  PUBMED    35263762
REFERENCE   2  (bases 1 to 9305)
  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 Cells.
            2022 Mar 9. pii: 6545973. doi: 10.1093/stmcls/sxac017."
COMMENT     Sequence Label: pSpCas9(BB)-2A-Puro(PX459)-GJA1 sgRNA
FEATURES             Location/Qualifiers
     source          1..9305
                     /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"
     misc_RNA        271..346
                     /label=gRNA scaffold
                     /note="guide RNA scaffold for the Streptococcus pyogenes 
                     CRISPR/Cas9 system"
     enhancer        443..728
                     /label=CMV enhancer
                     /note="human cytomegalovirus immediate early enhancer; 
                     contains an 18-bp deletion relative to the standard CMV 
                     enhancer"
     intron          1136..1363
                     /label=hybrid intron
                     /note="hybrid between chicken beta-actin (CBA) and minute 
                     virus of mice (MMV) introns (Gray et al., 2011)"
     regulatory      1375..1384
                     /label=Kozak sequence
                     /note="vertebrate consensus sequence for strong initiation 
                     of translation (Kozak, 1987)"
                     /regulatory_class="other"
     CDS             1384..1449
                     /codon_start=1
                     /product="three tandem FLAG(R) epitope tags, followed by an
                     enterokinase cleavage site"
                     /label=3xFLAG
                     /translation="DYKDHDGDYKDHDIDYKDDDDK"
     CDS             1456..1476
                     /codon_start=1
                     /product="nuclear localization signal of SV40 (simian virus
                     40) large T antigen"
                     /label=SV40 NLS
                     /translation="PKKKRKV"
     CDS             1501..5601
                     /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             1501..5601
                     /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             5602..5649
                     /codon_start=1
                     /product="bipartite nuclear localization signal from 
                     nucleoplasmin"
                     /label=nucleoplasmin NLS
                     /translation="KRPAATKKAGQAKKKK"
     CDS             5665..5718
                     /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             5719..6318
                     /codon_start=1
                     /gene="pac from Streptomyces alboniger"
                     /product="puromycin N-acetyltransferase"
                     /label=PuroR
                     /note="confers resistance to puromycin"
                     /translation="MTEYKPTVRLATRDDVPRAVRTLAAAFADYPATRHTVDPDRHIER
                     VTELQELFLTRVGLDIGKVWVADDGAAVAVWTTPESVEAGAVFAEIGPRMAELSGSRLA
                     AQQQMEGLLAPHRPKEPAWFLATVGVSPDHQGKGLGSAVVLPGVEAAERAGVPAFLETS
                     APHNLPFYERLGFTVTADVEVPEGPRTWCMTRKPGA"
     primer_bind     complement(5719..5738)
                     /label=Puro-R
                     /note="Puromycin resistance gene, reverse primer. Also 
                     called puro-variant-R"
     primer_bind     complement(6346..6363)
                     /label=BGH-rev
                     /note="Bovine growth hormone terminator, reverse primer. 
                     Also called BGH reverse"
     polyA_signal    6352..6559
                     /label=bGH poly(A) signal
                     /note="bovine growth hormone polyadenylation signal"
     repeat_region   6568..6708
                     /label=AAV2 ITR
                     /note="inverted terminal repeat of adeno-associated virus 
                     serotype 2"
     repeat_region   6568..6697
                     /label=AAV2 ITR
     rep_origin      6783..7238
                     /direction=RIGHT
                     /label=f1 ori
                     /note="f1 bacteriophage origin of replication; arrow 
                     indicates direction of (+) strand synthesis"
     primer_bind     complement(6870..6889)
                     /label=F1ori-R
                     /note="F1 origin, reverse primer"
     primer_bind     7080..7101
                     /label=F1ori-F
                     /note="F1 origin, forward primer"
     primer_bind     complement(7255..7274)
                     /label=pRS-marker
                     /note="pRS vectors, use to sequence yeast selectable 
                     marker"
     primer_bind     7374..7396
                     /label=pGEX 3'
                     /note="pGEX vectors, reverse primer"
     primer_bind     complement(7434..7452)
                     /label=pBRforEco
                     /note="pBR322 vectors, upsteam of EcoRI site, forward 
                     primer"
     promoter        7520..7624
                     /gene="bla"
                     /label=AmpR promoter
     CDS             join(7625..7693,7694..8485)
                     /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(7843..7862)
                     /label=Amp-R
                     /note="Ampicillin resistance gene, reverse primer"
     rep_origin      8656..9244
                     /direction=RIGHT
                     /label=ori
                     /note="high-copy-number ColE1/pMB1/pBR322/pUC origin of 
                     replication"
     primer_bind     9145..9164
                     /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 aagcctactc aactgctgga gttttagagc tagaaatagc aagttaaaat
      301 aaggctagtc cgttatcaac ttgaaaaagt ggcaccgagt cggtgctttt ttgttttaga
      361 gctagaaata gcaagttaaa ataaggctag tccgttttta gcgcgtgcgc caattctgca
      421 gacaaatggc tctagaggta cccgttacat aacttacggt aaatggcccg cctggctgac
      481 cgcccaacga cccccgccca ttgacgtcaa tagtaacgcc aatagggact ttccattgac
      541 gtcaatgggt ggagtattta cggtaaactg cccacttggc agtacatcaa gtgtatcata
      601 tgccaagtac gccccctatt gacgtcaatg acggtaaatg gcccgcctgg cattgtgccc
      661 agtacatgac cttatgggac tttcctactt ggcagtacat ctacgtatta gtcatcgcta
      721 ttaccatggt cgaggtgagc cccacgttct gcttcactct ccccatctcc cccccctccc
      781 cacccccaat tttgtattta tttatttttt aattattttg tgcagcgatg ggggcggggg
      841 gggggggggg gggcgccmcg gcgggggggg gggggggggg gggggggggg gggggggggg
      901 gggggggggg gggggggggg gggggggggg gggggggggg gggggggggg gggggggggg
      961 gggggggggg gggggggggn gggggggggg gggggggggg gggggggrgg ggsggggcgg
     1021 ggcgaggcgg agaggtgcgg cggcagccaa tcagagcggc gcgctccgaa agtttccttt
     1081 tatggcgagg cggcggcggc ggcggcccta taaaaagcga agcgcgcggc gggcgggagt
     1141 cgctgcgcgc tgccttcgcc ccgtgccccg ctccgccgcc gcctcgcgcc gcccgccccg
     1201 gctctgactg accgcgttac tcccacaggt gagcgggcgg gacggccctt ctcctccggg
     1261 ctgtaattag ctgagcaaga ggtaagggtt taagggatgg ttggttggtg gggtattaat
     1321 gtttaattac ctggagcacc tgcctgaaat cacttttttt caggttggac cggtgccacc
     1381 atggactata aggaccacga cggagactac aaggatcatg atattgatta caaagacgat
     1441 gacgataaga tggccccaaa gaagaagcgg aaggtcggta tccacggagt cccagcagcc
     1501 gacaagaagt acagcatcgg cctggacatc ggcaccaact ctgtgggctg ggccgtgatc
     1561 accgacgagt acaaggtgcc cagcaagaaa ttcaaggtgc tgggcaacac cgaccggcac
     1621 agcatcaaga agaacctgat cggagccctg ctgttcgaca gcggcgaaac agccgaggcc
     1681 acccggctga agagaaccgc cagaagaaga tacaccagac ggaagaaccg gatctgctat
     1741 ctgcaagaga tcttcagcaa cgagatggcc aaggtggacg acagcttctt ccacagactg
     1801 gaagagtcct tcctggtgga agaggataag aagcacgagc ggcaccccat cttcggcaac
     1861 atcgtggacg aggtggccta ccacgagaag taccccacca tctaccacct gagaaagaaa
     1921 ctggtggaca gcaccgacaa ggccgacctg cggctgatct atctggccct ggcccacatg
     1981 atcaagttcc ggggccactt cctgatcgag ggcgacctga accccgacaa cagcgacgtg
     2041 gacaagctgt tcatccagct ggtgcagacc tacaaccagc tgttcgagga aaaccccatc
     2101 aacgccagcg gcgtggacgc caaggccatc ctgtctgcca gactgagcaa gagcagacgg
     2161 ctggaaaatc tgatcgccca gctgcccggc gagaagaaga atggcctgtt cggaaacctg
     2221 attgccctga gcctgggcct gacccccaac ttcaagagca acttcgacct ggccgaggat
     2281 gccaaactgc agctgagcaa ggacacctac gacgacgacc tggacaacct gctggcccag
     2341 atcggcgacc agtacgccga cctgtttctg gccgccaaga acctgtccga cgccatcctg
     2401 ctgagcgaca tcctgagagt gaacaccgag atcaccaagg cccccctgag cgcctctatg
     2461 atcaagagat acgacgagca ccaccaggac ctgaccctgc tgaaagctct cgtgcggcag
     2521 cagctgcctg agaagtacaa agagattttc ttcgaccaga gcaagaacgg ctacgccggc
     2581 tacattgacg gcggagccag ccaggaagag ttctacaagt tcatcaagcc catcctggaa
     2641 aagatggacg gcaccgagga actgctcgtg aagctgaaca gagaggacct gctgcggaag
     2701 cagcggacct tcgacaacgg cagcatcccc caccagatcc acctgggaga gctgcacgcc
     2761 attctgcggc ggcaggaaga tttttaccca ttcctgaagg acaaccggga aaagatcgag
     2821 aagatcctga ccttccgcat cccctactac gtgggccctc tggccagggg aaacagcaga
     2881 ttcgcctgga tgaccagaaa gagcgaggaa accatcaccc cctggaactt cgaggaagtg
     2941 gtggacaagg gcgcttccgc ccagagcttc atcgagcgga tgaccaactt cgataagaac
     3001 ctgcccaacg agaaggtgct gcccaagcac agcctgctgt acgagtactt caccgtgtat
     3061 aacgagctga ccaaagtgaa atacgtgacc gagggaatga gaaagcccgc cttcctgagc
     3121 ggcgagcaga aaaaggccat cgtggacctg ctgttcaaga ccaaccggaa agtgaccgtg
     3181 aagcagctga aagaggacta cttcaagaaa atcgagtgct tcgactccgt ggaaatctcc
     3241 ggcgtggaag atcggttcaa cgcctccctg ggcacatacc acgatctgct gaaaattatc
     3301 aaggacaagg acttcctgga caatgaggaa aacgaggaca ttctggaaga tatcgtgctg
     3361 accctgacac tgtttgagga cagagagatg atcgaggaac ggctgaaaac ctatgcccac
     3421 ctgttcgacg acaaagtgat gaagcagctg aagcggcgga gatacaccgg ctggggcagg
     3481 ctgagccgga agctgatcaa cggcatccgg gacaagcagt ccggcaagac aatcctggat
     3541 ttcctgaagt ccgacggctt cgccaacaga aacttcatgc agctgatcca cgacgacagc
     3601 ctgaccttta aagaggacat ccagaaagcc caggtgtccg gccagggcga tagcctgcac
     3661 gagcacattg ccaatctggc cggcagcccc gccattaaga agggcatcct gcagacagtg
     3721 aaggtggtgg acgagctcgt gaaagtgatg ggccggcaca agcccgagaa catcgtgatc
     3781 gaaatggcca gagagaacca gaccacccag aagggacaga agaacagccg cgagagaatg
     3841 aagcggatcg aagagggcat caaagagctg ggcagccaga tcctgaaaga acaccccgtg
     3901 gaaaacaccc agctgcagaa cgagaagctg tacctgtact acctgcagaa tgggcgggat
     3961 atgtacgtgg accaggaact ggacatcaac cggctgtccg actacgatgt ggaccatatc
     4021 gtgcctcaga gctttctgaa ggacgactcc atcgacaaca aggtgctgac cagaagcgac
     4081 aagaaccggg gcaagagcga caacgtgccc tccgaagagg tcgtgaagaa gatgaagaac
     4141 tactggcggc agctgctgaa cgccaagctg attacccaga gaaagttcga caatctgacc
     4201 aaggccgaga gaggcggcct gagcgaactg gataaggccg gcttcatcaa gagacagctg
     4261 gtggaaaccc ggcagatcac aaagcacgtg gcacagatcc tggactcccg gatgaacact
     4321 aagtacgacg agaatgacaa gctgatccgg gaagtgaaag tgatcaccct gaagtccaag
     4381 ctggtgtccg atttccggaa ggatttccag ttttacaaag tgcgcgagat caacaactac
     4441 caccacgccc acgacgccta cctgaacgcc gtcgtgggaa ccgccctgat caaaaagtac
     4501 cctaagctgg aaagcgagtt cgtgtacggc gactacaagg tgtacgacgt gcggaagatg
     4561 atcgccaaga gcgagcagga aatcggcaag gctaccgcca agtacttctt ctacagcaac
     4621 atcatgaact ttttcaagac cgagattacc ctggccaacg gcgagatccg gaagcggcct
     4681 ctgatcgaga caaacggcga aaccggggag atcgtgtggg ataagggccg ggattttgcc
     4741 accgtgcgga aagtgctgag catgccccaa gtgaatatcg tgaaaaagac cgaggtgcag
     4801 acaggcggct tcagcaaaga gtctatcctg cccaagagga acagcgataa gctgatcgcc
     4861 agaaagaagg actgggaccc taagaagtac ggcggcttcg acagccccac cgtggcctat
     4921 tctgtgctgg tggtggccaa agtggaaaag ggcaagtcca agaaactgaa gagtgtgaaa
     4981 gagctgctgg ggatcaccat catggaaaga agcagcttcg agaagaatcc catcgacttt
     5041 ctggaagcca agggctacaa agaagtgaaa aaggacctga tcatcaagct gcctaagtac
     5101 tccctgttcg agctggaaaa cggccggaag agaatgctgg cctctgccgg cgaactgcag
     5161 aagggaaacg aactggccct gccctccaaa tatgtgaact tcctgtacct ggccagccac
     5221 tatgagaagc tgaagggctc ccccgaggat aatgagcaga aacagctgtt tgtggaacag
     5281 cacaagcact acctggacga gatcatcgag cagatcagcg agttctccaa gagagtgatc
     5341 ctggccgacg ctaatctgga caaagtgctg tccgcctaca acaagcaccg ggataagccc
     5401 atcagagagc aggccgagaa tatcatccac ctgtttaccc tgaccaatct gggagcccct
     5461 gccgccttca agtactttga caccaccatc gaccggaaga ggtacaccag caccaaagag
     5521 gtgctggacg ccaccctgat ccaccagagc atcaccggcc tgtacgagac acggatcgac
     5581 ctgtctcagc tgggaggcga caaaaggccg gcggccacga aaaaggccgg ccaggcaaaa
     5641 aagaaaaagg aattcggcag tggagagggc agaggaagtc tgctaacatg cggtgacgtc
     5701 gaggagaatc ctggcccaat gaccgagtac aagcccacgg tgcgcctcgc cacccgcgac
     5761 gacgtcccca gggccgtacg caccctcgcc gccgcgttcg ccgactaccc cgccacgcgc
     5821 cacaccgtcg atccggaccg ccacatcgag cgggtcaccg agctgcaaga actcttcctc
     5881 acgcgcgtcg ggctcgacat cggcaaggtg tgggtcgcgg acgacggcgc cgcggtggcg
     5941 gtctggacca cgccggagag cgtcgaagcg ggggcggtgt tcgccgagat cggcccgcgc
     6001 atggccgagt tgagcggttc ccggctggcc gcgcagcaac agatggaagg cctcctggcg
     6061 ccgcaccggc ccaaggagcc cgcgtggttc ctggccaccg tcggcgtctc gcccgaccac
     6121 cagggcaagg gtctgggcag cgccgtcgtg ctccccggag tggaggcggc cgagcgcgcc
     6181 ggggtgcccg ccttcctgga gacctccgcg ccccacaacc tccccttcta cgagcggctc
     6241 ggcttcaccg tcaccgccga cgtcgaggtg cccgaaggac cgcgcacctg gtgcatgacc
     6301 cgcaagcccg gtgcctgaga attctaacta gagctcgctg atcagcctcg actgtgcctt
     6361 ctagttgcca gccatctgtt gtttgcccct cccccgtgcc ttccttgacc ctggaaggtg
     6421 ccactcccac tgtcctttcc taataaaatg aggaaattgc atcgcattgt ctgagtaggt
     6481 gtcattctat tctggggggt ggggtggggc aggacagcaa gggggaggat tgggaagaga
     6541 atagcaggca tgctggggag cggccgcagg aacccctagt gatggagttg gccactccct
     6601 ctctgcgcgc tcgctcgctc actgaggccg ggcgaccaaa ggtcgcccga cgcccgggct
     6661 ttgcccgggc ggcctcagtg agcgagcgag cgcgcagctg cctgcagggg cgcctgatgc
     6721 ggtattttct ccttacgcat ctgtgcggta tttcacaccg catacgtcaa agcaaccata
     6781 gtacgcgccc tgtagcggcg cattaagcgc ggcgggtgtg gtggttacgc gcagcgtgac
     6841 cgctacactt gccagcgcct tagcgcccgc tcctttcgct ttcttccctt cctttctcgc
     6901 cacgttcgcc ggctttcccc gtcaagctct aaatcggggg ctccctttag ggttccgatt
     6961 tagtgcttta cggcacctcg accccaaaaa acttgatttg ggtgatggtt cacgtagtgg
     7021 gccatcgccc tgatagacgg tttttcgccc tttgacgttg gagtccacgt tctttaatag
     7081 tggactcttg ttccaaactg gaacaacact caactctatc tcgggctatt cttttgattt
     7141 ataagggatt ttgccgattt cggtctattg gttaaaaaat gagctgattt aacaaaaatt
     7201 taacgcgaat tttaacaaaa tattaacgtt tacaatttta tggtgcactc tcagtacaat
     7261 ctgctctgat gccgcatagt taagccagcc ccgacacccg ccaacacccg ctgacgcgcc
     7321 ctgacgggct tgtctgctcc cggcatccgc ttacagacaa gctgtgaccg tctccgggag
     7381 ctgcatgtgt cagaggtttt caccgtcatc accgaaacgc gcgagacgaa agggcctcgt
     7441 gatacgccta tttttatagg ttaatgtcat gataataatg gtttcttaga cgtcaggtgg
     7501 cacttttcgg ggaaatgtgc gcggaacccc tatttgttta tttttctaaa tacattcaaa
     7561 tatgtatccg ctcatgagac aataaccctg ataaatgctt caataatatt gaaaaaggaa
     7621 gagtatgagt attcaacatt tccgtgtcgc ccttattccc ttttttgcgg cattttgcct
     7681 tcctgttttt gctcacccag aaacgctggt gaaagtaaaa gatgctgaag atcagttggg
     7741 tgcacgagtg ggttacatcg aactggatct caacagcggt aagatccttg agagttttcg
     7801 ccccgaagaa cgttttccaa tgatgagcac ttttaaagtt ctgctatgtg gcgcggtatt
     7861 atcccgtatt gacgccgggc aagagcaact cggtcgccgc atacactatt ctcagaatga
     7921 cttggttgag tactcaccag tcacagaaaa gcatcttacg gatggcatga cagtaagaga
     7981 attatgcagt gctgccataa ccatgagtga taacactgcg gccaacttac ttctgacaac
     8041 gatcggagga ccgaaggagc taaccgcttt tttgcacaac atgggggatc atgtaactcg
     8101 ccttgatcgt tgggaaccgg agctgaatga agccatacca aacgacgagc gtgacaccac
     8161 gatgcctgta gcaatggcaa caacgttgcg caaactatta actggcgaac tacttactct
     8221 agcttcccgg caacaattaa tagactggat ggaggcggat aaagttgcag gaccacttct
     8281 gcgctcggcc cttccggctg gctggtttat tgctgataaa tctggagccg gtgagcgtgg
     8341 aagccgcggt atcattgcag cactggggcc agatggtaag ccctcccgta tcgtagttat
     8401 ctacacgacg gggagtcagg caactatgga tgaacgaaat agacagatcg ctgagatagg
     8461 tgcctcactg attaagcatt ggtaactgtc agaccaagtt tactcatata tactttagat
     8521 tgatttaaaa cttcattttt aatttaaaag gatctaggtg aagatccttt ttgataatct
     8581 catgaccaaa atcccttaac gtgagttttc gttccactga gcgtcagacc ccgtagaaaa
     8641 gatcaaagga tcttcttgag atcctttttt tctgcgcgta atctgctgct tgcaaacaaa
     8701 aaaaccaccg ctaccagcgg tggtttgttt gccggatcaa gagctaccaa ctctttttcc
     8761 gaaggtaact ggcttcagca gagcgcagat accaaatact gttcttctag tgtagccgta
     8821 gttaggccac cacttcaaga actctgtagc accgcctaca tacctcgctc tgctaatcct
     8881 gttaccagtg gctgctgcca gtggcgataa gtcgtgtctt accgggttgg actcaagacg
     8941 atagttaccg gataaggcgc agcggtcggg ctgaacgggg ggttcgtgca cacagcccag
     9001 cttggagcga acgacctaca ccgaactgag atacctacag cgtgagctat gagaaagcgc
     9061 cacgcttccc gaagggagaa aggcggacag gtatccggta agcggcaggg tcggaacagg
     9121 agagcgcacg agggagcttc cagggggaaa cgcctggtat ctttatagtc ctgtcgggtt
     9181 tcgccacctc tgacttgagc gtcgattttt gtgatgctcg tcaggggggc ggagcctatg
     9241 gaaaaacgcc agcaacgcgg cctttttacg gttcctggcc ttttgctggc cttttgctca
     9301 catgt
//
