LOCUS       sequence-328107-       10222 bp    DNA     circular SYN 15-DEC-2025
DEFINITION  Empty backbone for cloning gRNA sequences for DNA targeting. 
            Contains an T2A linked H2B GFP nuclear reporter..
ACCESSION   .
VERSION     .
KEYWORDS    .
SOURCE      synthetic DNA construct
  ORGANISM  synthetic DNA construct
REFERENCE   1  (bases 1 to 10222)
  AUTHORS   Kaufman ML, Goodson NB, Park KU, Schwanke M, Office E, Schneider SR,
            Abraham J, Hensley A, Jones KL, Brzezinski JA
  TITLE     Initiation of Otx2 expression in the developing mouse retina 
            requires a unique enhancer and either Ascl1 or Neurog2 activity.
  JOURNAL   Development. 2021 Jun 15;148(12). pii: 269198. doi: 
            10.1242/dev.199399. Epub 2021 Jun 18.
  PUBMED    34143204
REFERENCE   2  (bases 1 to 10222)
  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: 
            "Development."; date: "2021-06-15"; volume: "148(12). pii: 269198. 
            doi"; pages: " 10.1242/dev.199399. Epub 2021 Jun 18"
COMMENT     Sequence Label: pX458-Ef1a-Cas9-H2B-GFP
FEATURES             Location/Qualifiers
     source          1..10222
                     /mol_type="other DNA"
                     /organism="synthetic DNA construct"
     promoter        122..1304
                     /label=EF-1-alpha promoter
                     /note="strong constitutive promoter for human elongation 
                     factor EF-1-alpha"
     intron          353..1295
                     /label=EF-1-alpha intron A
                     /note="intron upstream of the start codon of human 
                     EF-1-alpha"
     primer_bind     1252..1272
                     /label=EF1a-F
                     /note="Human elongation factor-1a promoter, forward primer"
     regulatory      1310..1319
                     /label=Kozak sequence
                     /note="vertebrate consensus sequence for strong initiation 
                     of translation (Kozak, 1987)"
                     /regulatory_class="other"
     CDS             1319..1384
                     /codon_start=1
                     /product="three tandem FLAG(R) epitope tags, followed by an
                     enterokinase cleavage site"
                     /label=3xFLAG
                     /translation="DYKDHDGDYKDHDIDYKDDDDK"
     CDS             1391..1411
                     /codon_start=1
                     /product="nuclear localization signal of SV40 (simian virus
                     40) large T antigen"
                     /label=SV40 NLS
                     /translation="PKKKRKV"
     CDS             1436..5536
                     /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             1436..5536
                     /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             5537..5584
                     /codon_start=1
                     /product="bipartite nuclear localization signal from 
                     nucleoplasmin"
                     /label=nucleoplasmin NLS
                     /translation="KRPAATKKAGQAKKKK"
     CDS             5600..5653
                     /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             5654..6031
                     /codon_start=1
                     /gene="HIST1H2BB"
                     /product="human histone H2B"
                     /label=H2B
                     /translation="MPEPSKSAPAPKKGSKKAITKAQKKDGKKRKRSRKESYSIYVYKV
                     LKQVHPDTGISSKAMGIMNSFVNDIFERIAGEASRLAHYNKRSTITSREIQTAVRLLLP
                     GELAKHAVSEGTKAVTKYTSSK"
     regulatory      6044..6053
                     /label=Kozak sequence
                     /note="vertebrate consensus sequence for strong initiation 
                     of translation (Kozak, 1987)"
                     /regulatory_class="other"
     CDS             join(6050..6052,6053..6055,6056..6766)
                     /codon_start=1
                     /product="enhanced GFP"
                     /label=EGFP
                     /translation="MVSKGEELFTGVVPILVELDGDVNGHKFSVSGEGEGDATYGKLTL
                     KFICTTGKLPVPWPTLVTTLTYGVQCFSRYPDHMKQHDFFKSAMPEGYVQERTIFFKDD
                     GNYKTRAEVKFEGDTLVNRIELKGIDFKEDGNILGHKLEYNYNSHNVYIMADKQKNGIK
                     VNFKIRHNIEDGSVQLADHYQQNTPIGDGPVLLPDNHYLSTQSALSKDPNEKRDHMVLL
                     EFVTAAGITLGMDELYK"
     CDS             join(6050..6052,6053..6055,6056..6766)
                     /codon_start=1
                     /product="the original enhanced GFP (Yang et al., 1996)"
                     /label=EGFP
                     /note="mammalian codon-optimized"
                     /translation="MVSKGEELFTGVVPILVELDGDVNGHKFSVSGEGEGDATYGKLTL
                     KFICTTGKLPVPWPTLVTTLTYGVQCFSRYPDHMKQHDFFKSAMPEGYVQERTIFFKDD
                     GNYKTRAEVKFEGDTLVNRIELKGIDFKEDGNILGHKLEYNYNSHNVYIMADKQKNGIK
                     VNFKIRHNIEDGSVQLADHYQQNTPIGDGPVLLPDNHYLSTQSALSKDPNEKRDHMVLL
                     EFVTAAGITLGMDELYK"
     primer_bind     complement(6356..6375)
                     /label=EXFP-R
                     /note="For distinguishing EGFP variants, reverse primer"
     primer_bind     6703..6724
                     /label=EGFP-C
                     /note="EGFP, forward primer"
     primer_bind     complement(6794..6811)
                     /label=BGH-rev
                     /note="Bovine growth hormone terminator, reverse primer. 
                     Also called BGH reverse"
     polyA_signal    6800..7007
                     /label=bGH poly(A) signal
                     /note="bovine growth hormone polyadenylation signal"
     repeat_region   7016..7156
                     /label=AAV2 ITR
                     /note="inverted terminal repeat of adeno-associated virus 
                     serotype 2"
     repeat_region   7016..7145
                     /label=AAV2 ITR
     rep_origin      7231..7686
                     /direction=RIGHT
                     /label=f1 ori
                     /note="f1 bacteriophage origin of replication; arrow 
                     indicates direction of (+) strand synthesis"
     primer_bind     complement(7318..7337)
                     /label=F1ori-R
                     /note="F1 origin, reverse primer"
     primer_bind     7528..7549
                     /label=F1ori-F
                     /note="F1 origin, forward primer"
     primer_bind     complement(7703..7722)
                     /label=pRS-marker
                     /note="pRS vectors, use to sequence yeast selectable 
                     marker"
     primer_bind     7822..7844
                     /label=pGEX 3'
                     /note="pGEX vectors, reverse primer"
     primer_bind     complement(7882..7900)
                     /label=pBRforEco
                     /note="pBR322 vectors, upsteam of EcoRI site, forward 
                     primer"
     promoter        7968..8072
                     /gene="bla"
                     /label=AmpR promoter
     CDS             join(8073..8141,8142..8933)
                     /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(8291..8310)
                     /label=Amp-R
                     /note="Ampicillin resistance gene, reverse primer"
     rep_origin      9104..9692
                     /direction=RIGHT
                     /label=ori
                     /note="high-copy-number ColE1/pMB1/pBR322/pUC origin of 
                     replication"
     primer_bind     9593..9612
                     /label=pBR322ori-F
                     /note="pBR322 origin, forward primer"
     promoter        9754..10002
                     /label=U6 promoter
                     /note="RNA polymerase III promoter for human U6 snRNA 
                     (Domitrovich & Kunkel, 2003)"
     primer_bind     9754..9774
                     /label=hU6-F
                     /note="Human U6 promoter, forward primer"
     primer_bind     9925..9944
                     /label=LKO.1 5'
                     /note="Human U6 promoter, forward primer"
     misc_RNA        10021..10096
                     /label=gRNA scaffold
                     /note="guide RNA scaffold for the Streptococcus pyogenes 
                     CRISPR/Cas9 system"
ORIGIN
        1 cttggggaat cccagggacc gtcgttaaac tcccactaac gtagaaccca gagatcgctg
       61 cgttcccgcc ccctcacccg cccgctctcg tcatcactga ggtggagaag agcatgcgtg
      121 aggctccggt gcccgtcagt gggcagagcg cacatcgccc acagtccccg agaagttggg
      181 gggaggggtc ggcaattgaa ccggtgccta gagaaggtgg cgcggggtaa actgggaaag
      241 tgatgtcgtg tactggctcc gcctttttcc cgagggtggg ggagaaccgt atataagtgc
      301 agtagtcgcc gtgaacgttc tttttcgcaa cgggtttgcc gccagaacac aggtaagtgc
      361 cgtgtgtggt tcccgcgggc ctggcctctt tacgggttat ggcccttgcg tgccttgaat
      421 tacttccacg cccctggctg cagtacgtga ttcttgatcc cgagcttcgg gttggaagtg
      481 ggtgggagag ttcgaggcct tgcgcttaag gagccccttc gcctcgtgct tgagttgagg
      541 cctggcttgg gcgctggggc cgccgcgtgc gaatctggtg gcaccttcgc gcctgtctcg
      601 ctgctttcga taagtctcta gccatttaaa atttttgatg acctgctgcg acgctttttt
      661 tctggcaaga tagtcttgta aatgcgggcc aagatctgca cactggtatt tcggtttttg
      721 gggccgcggg cggcgacggg gcccgtgcgt cccagcgcac atgttcggcg aggcggggcc
      781 tgcgagcgcg gccaccgaga atcggacggg ggtagtctca agctggccgg cctgctctgg
      841 tgcctggcct cgcgccgccg tgtatcgccc cgccctgggc ggcaaggctg gcccggtcgg
      901 caccagttgc gtgagcggaa agatggccgc ttcccggccc tgctgcaggg agctcaaaat
      961 ggaggacgcg gcgctcggga gagcgggcgg gtgagtcacc cacacaaagg aaaagggcct
     1021 ttccgtcctc agccgtcgct tcatgtgact ccacggagta ccgggcgccg tccaggcacc
     1081 tcgattagtt ctcgagcttt tggagtacgt cgtctttagg ttggggggag gggttttatg
     1141 cgatggagtt tccccacact gagtgggtgg agactgaagt taggccagct tggcacttga
     1201 tgtaattctc cttggaattt gccctttttg agtttggatc ttggttcatt ctcaagcctc
     1261 agacagtggt tcaaagtttt tttcttccat ttcaggtgtc gtgaccggtg ccaccatgga
     1321 ctataaggac cacgacggag actacaagga tcatgatatt gattacaaag acgatgacga
     1381 taagatggcc ccaaagaaga agcggaaggt cggtatccac ggagtcccag cagccgacaa
     1441 gaagtacagc atcggcctgg acatcggcac caactctgtg ggctgggccg tgatcaccga
     1501 cgagtacaag gtgcccagca agaaattcaa ggtgctgggc aacaccgacc ggcacagcat
     1561 caagaagaac ctgatcggag ccctgctgtt cgacagcggc gaaacagccg aggccacccg
     1621 gctgaagaga accgccagaa gaagatacac cagacggaag aaccggatct gctatctgca
     1681 agagatcttc agcaacgaga tggccaaggt ggacgacagc ttcttccaca gactggaaga
     1741 gtccttcctg gtggaagagg ataagaagca cgagcggcac cccatcttcg gcaacatcgt
     1801 ggacgaggtg gcctaccacg agaagtaccc caccatctac cacctgagaa agaaactggt
     1861 ggacagcacc gacaaggccg acctgcggct gatctatctg gccctggccc acatgatcaa
     1921 gttccggggc cacttcctga tcgagggcga cctgaacccc gacaacagcg acgtggacaa
     1981 gctgttcatc cagctggtgc agacctacaa ccagctgttc gaggaaaacc ccatcaacgc
     2041 cagcggcgtg gacgccaagg ccatcctgtc tgccagactg agcaagagca gacggctgga
     2101 aaatctgatc gcccagctgc ccggcgagaa gaagaatggc ctgttcggaa acctgattgc
     2161 cctgagcctg ggcctgaccc ccaacttcaa gagcaacttc gacctggccg aggatgccaa
     2221 actgcagctg agcaaggaca cctacgacga cgacctggac aacctgctgg cccagatcgg
     2281 cgaccagtac gccgacctgt ttctggccgc caagaacctg tccgacgcca tcctgctgag
     2341 cgacatcctg agagtgaaca ccgagatcac caaggccccc ctgagcgcct ctatgatcaa
     2401 gagatacgac gagcaccacc aggacctgac cctgctgaaa gctctcgtgc ggcagcagct
     2461 gcctgagaag tacaaagaga ttttcttcga ccagagcaag aacggctacg ccggctacat
     2521 tgacggcgga gccagccagg aagagttcta caagttcatc aagcccatcc tggaaaagat
     2581 ggacggcacc gaggaactgc tcgtgaagct gaacagagag gacctgctgc ggaagcagcg
     2641 gaccttcgac aacggcagca tcccccacca gatccacctg ggagagctgc acgccattct
     2701 gcggcggcag gaagattttt acccattcct gaaggacaac cgggaaaaga tcgagaagat
     2761 cctgaccttc cgcatcccct actacgtggg ccctctggcc aggggaaaca gcagattcgc
     2821 ctggatgacc agaaagagcg aggaaaccat caccccctgg aacttcgagg aagtggtgga
     2881 caagggcgct tccgcccaga gcttcatcga gcggatgacc aacttcgata agaacctgcc
     2941 caacgagaag gtgctgccca agcacagcct gctgtacgag tacttcaccg tgtataacga
     3001 gctgaccaaa gtgaaatacg tgaccgaggg aatgagaaag cccgccttcc tgagcggcga
     3061 gcagaaaaag gccatcgtgg acctgctgtt caagaccaac cggaaagtga ccgtgaagca
     3121 gctgaaagag gactacttca agaaaatcga gtgcttcgac tccgtggaaa tctccggcgt
     3181 ggaagatcgg ttcaacgcct ccctgggcac ataccacgat ctgctgaaaa ttatcaagga
     3241 caaggacttc ctggacaatg aggaaaacga ggacattctg gaagatatcg tgctgaccct
     3301 gacactgttt gaggacagag agatgatcga ggaacggctg aaaacctatg cccacctgtt
     3361 cgacgacaaa gtgatgaagc agctgaagcg gcggagatac accggctggg gcaggctgag
     3421 ccggaagctg atcaacggca tccgggacaa gcagtccggc aagacaatcc tggatttcct
     3481 gaagtccgac ggcttcgcca acagaaactt catgcagctg atccacgacg acagcctgac
     3541 ctttaaagag gacatccaga aagcccaggt gtccggccag ggcgatagcc tgcacgagca
     3601 cattgccaat ctggccggca gccccgccat taagaagggc atcctgcaga cagtgaaggt
     3661 ggtggacgag ctcgtgaaag tgatgggccg gcacaagccc gagaacatcg tgatcgaaat
     3721 ggccagagag aaccagacca cccagaaggg acagaagaac agccgcgaga gaatgaagcg
     3781 gatcgaagag ggcatcaaag agctgggcag ccagatcctg aaagaacacc ccgtggaaaa
     3841 cacccagctg cagaacgaga agctgtacct gtactacctg cagaatgggc gggatatgta
     3901 cgtggaccag gaactggaca tcaaccggct gtccgactac gatgtggacc atatcgtgcc
     3961 tcagagcttt ctgaaggacg actccatcga caacaaggtg ctgaccagaa gcgacaagaa
     4021 ccggggcaag agcgacaacg tgccctccga agaggtcgtg aagaagatga agaactactg
     4081 gcggcagctg ctgaacgcca agctgattac ccagagaaag ttcgacaatc tgaccaaggc
     4141 cgagagaggc ggcctgagcg aactggataa ggccggcttc atcaagagac agctggtgga
     4201 aacccggcag atcacaaagc acgtggcaca gatcctggac tcccggatga acactaagta
     4261 cgacgagaat gacaagctga tccgggaagt gaaagtgatc accctgaagt ccaagctggt
     4321 gtccgatttc cggaaggatt tccagtttta caaagtgcgc gagatcaaca actaccacca
     4381 cgcccacgac gcctacctga acgccgtcgt gggaaccgcc ctgatcaaaa agtaccctaa
     4441 gctggaaagc gagttcgtgt acggcgacta caaggtgtac gacgtgcgga agatgatcgc
     4501 caagagcgag caggaaatcg gcaaggctac cgccaagtac ttcttctaca gcaacatcat
     4561 gaactttttc aagaccgaga ttaccctggc caacggcgag atccggaagc ggcctctgat
     4621 cgagacaaac ggcgaaaccg gggagatcgt gtgggataag ggccgggatt ttgccaccgt
     4681 gcggaaagtg ctgagcatgc cccaagtgaa tatcgtgaaa aagaccgagg tgcagacagg
     4741 cggcttcagc aaagagtcta tcctgcccaa gaggaacagc gataagctga tcgccagaaa
     4801 gaaggactgg gaccctaaga agtacggcgg cttcgacagc cccaccgtgg cctattctgt
     4861 gctggtggtg gccaaagtgg aaaagggcaa gtccaagaaa ctgaagagtg tgaaagagct
     4921 gctggggatc accatcatgg aaagaagcag cttcgagaag aatcccatcg actttctgga
     4981 agccaagggc tacaaagaag tgaaaaagga cctgatcatc aagctgccta agtactccct
     5041 gttcgagctg gaaaacggcc ggaagagaat gctggcctct gccggcgaac tgcagaaggg
     5101 aaacgaactg gccctgccct ccaaatatgt gaacttcctg tacctggcca gccactatga
     5161 gaagctgaag ggctcccccg aggataatga gcagaaacag ctgtttgtgg aacagcacaa
     5221 gcactacctg gacgagatca tcgagcagat cagcgagttc tccaagagag tgatcctggc
     5281 cgacgctaat ctggacaaag tgctgtccgc ctacaacaag caccgggata agcccatcag
     5341 agagcaggcc gagaatatca tccacctgtt taccctgacc aatctgggag cccctgccgc
     5401 cttcaagtac tttgacacca ccatcgaccg gaagaggtac accagcacca aagaggtgct
     5461 ggacgccacc ctgatccacc agagcatcac cggcctgtac gagacacgga tcgacctgtc
     5521 tcagctggga ggcgacaaaa ggccggcggc cacgaaaaag gccggccagg caaaaaagaa
     5581 aaaggaattc ggcagtggag agggcagagg aagtctgcta acatgcggtg acgtcgagga
     5641 gaatcctggc ccaatgcctg aaccctctaa gtctgctcca gcccctaaaa agggttctaa
     5701 gaaggctatc actaaggcgc agaagaagga tggtaagaag cgtaagcgca gccgcaagga
     5761 gagctattct atctatgtgt acaaggttct gaagcaggtc caccccgaca ccggcatctc
     5821 atccaaggcc atggggatca tgaactcctt cgtcaacgac atcttcgagc gcatcgcggg
     5881 cgaggcttct cgcctggctc actacaataa gcgctcgacc atcacctcca gggagattca
     5941 gacggctgtg cgcctgctgc tgcctgggga gctggctaag catgctgtgt ccgagggcac
     6001 taaggcagtt accaagtaca ctagctctaa ggatccaccg gtcgccacca tggtgagcaa
     6061 gggcgaggag ctgttcaccg gggtggtgcc catcctggtc gagctggacg gcgacgtaaa
     6121 cggccacaag ttcagcgtgt ccggcgaggg cgagggcgat gccacctacg gcaagctgac
     6181 cctgaagttc atctgcacca ccggcaagct gcccgtgccc tggcccaccc tcgtgaccac
     6241 cctgacctac ggcgtgcagt gcttcagccg ctaccccgac cacatgaagc agcacgactt
     6301 cttcaagtcc gccatgcccg aaggctacgt ccaggagcgc accatcttct tcaaggacga
     6361 cggcaactac aagacccgcg ccgaggtgaa gttcgagggc gacaccctgg tgaaccgcat
     6421 cgagctgaag ggcatcgact tcaaggagga cggcaacatc ctggggcaca agctggagta
     6481 caactacaac agccacaacg tctatatcat ggccgacaag cagaagaacg gcatcaaggt
     6541 gaacttcaag atccgccaca acatcgagga cggcagcgtg cagctcgccg accactacca
     6601 gcagaacacc cccatcggcg acggccccgt gctgctgccc gacaaccact acctgagcac
     6661 ccagtccgcc ctgagcaaag accccaacga gaagcgcgat cacatggtcc tgctggagtt
     6721 cgtgaccgcc gccgggatca ctctcggcat ggacgagctg tacaaggaat tctaactaga
     6781 gctcgctgat cagcctcgac tgtgccttct agttgccagc catctgttgt ttgcccctcc
     6841 cccgtgcctt ccttgaccct ggaaggtgcc actcccactg tcctttccta ataaaatgag
     6901 gaaattgcat cgcattgtct gagtaggtgt cattctattc tggggggtgg ggtggggcag
     6961 gacagcaagg gggaggattg ggaagagaat agcaggcatg ctggggagcg gccgcaggaa
     7021 cccctagtga tggagttggc cactccctct ctgcgcgctc gctcgctcac tgaggccggg
     7081 cgaccaaagg tcgcccgacg cccgggcttt gcccgggcgg cctcagtgag cgagcgagcg
     7141 cgcagctgcc tgcaggggcg cctgatgcgg tattttctcc ttacgcatct gtgcggtatt
     7201 tcacaccgca tacgtcaaag caaccatagt acgcgccctg tagcggcgca ttaagcgcgg
     7261 cgggtgtggt ggttacgcgc agcgtgaccg ctacacttgc cagcgcctta gcgcccgctc
     7321 ctttcgcttt cttcccttcc tttctcgcca cgttcgccgg ctttccccgt caagctctaa
     7381 atcgggggct ccctttaggg ttccgattta gtgctttacg gcacctcgac cccaaaaaac
     7441 ttgatttggg tgatggttca cgtagtgggc catcgccctg atagacggtt tttcgccctt
     7501 tgacgttgga gtccacgttc tttaatagtg gactcttgtt ccaaactgga acaacactca
     7561 actctatctc gggctattct tttgatttat aagggatttt gccgatttcg gtctattggt
     7621 taaaaaatga gctgatttaa caaaaattta acgcgaattt taacaaaata ttaacgttta
     7681 caattttatg gtgcactctc agtacaatct gctctgatgc cgcatagtta agccagcccc
     7741 gacacccgcc aacacccgct gacgcgccct gacgggcttg tctgctcccg gcatccgctt
     7801 acagacaagc tgtgaccgtc tccgggagct gcatgtgtca gaggttttca ccgtcatcac
     7861 cgaaacgcgc gagacgaaag ggcctcgtga tacgcctatt tttataggtt aatgtcatga
     7921 taataatggt ttcttagacg tcaggtggca cttttcgggg aaatgtgcgc ggaaccccta
     7981 tttgtttatt tttctaaata cattcaaata tgtatccgct catgagacaa taaccctgat
     8041 aaatgcttca ataatattga aaaaggaaga gtatgagtat tcaacatttc cgtgtcgccc
     8101 ttattccctt ttttgcggca ttttgccttc ctgtttttgc tcacccagaa acgctggtga
     8161 aagtaaaaga tgctgaagat cagttgggtg cacgagtggg ttacatcgaa ctggatctca
     8221 acagcggtaa gatccttgag agttttcgcc ccgaagaacg ttttccaatg atgagcactt
     8281 ttaaagttct gctatgtggc gcggtattat cccgtattga cgccgggcaa gagcaactcg
     8341 gtcgccgcat acactattct cagaatgact tggttgagta ctcaccagtc acagaaaagc
     8401 atcttacgga tggcatgaca gtaagagaat tatgcagtgc tgccataacc atgagtgata
     8461 acactgcggc caacttactt ctgacaacga tcggaggacc gaaggagcta accgcttttt
     8521 tgcacaacat gggggatcat gtaactcgcc ttgatcgttg ggaaccggag ctgaatgaag
     8581 ccataccaaa cgacgagcgt gacaccacga tgcctgtagc aatggcaaca acgttgcgca
     8641 aactattaac tggcgaacta cttactctag cttcccggca acaattaata gactggatgg
     8701 aggcggataa agttgcagga ccacttctgc gctcggccct tccggctggc tggtttattg
     8761 ctgataaatc tggagccggt gagcgtggaa gccgcggtat cattgcagca ctggggccag
     8821 atggtaagcc ctcccgtatc gtagttatct acacgacggg gagtcaggca actatggatg
     8881 aacgaaatag acagatcgct gagataggtg cctcactgat taagcattgg taactgtcag
     8941 accaagttta ctcatatata ctttagattg atttaaaact tcatttttaa tttaaaagga
     9001 tctaggtgaa gatccttttt gataatctca tgaccaaaat cccttaacgt gagttttcgt
     9061 tccactgagc gtcagacccc gtagaaaaga tcaaaggatc ttcttgagat cctttttttc
     9121 tgcgcgtaat ctgctgcttg caaacaaaaa aaccaccgct accagcggtg gtttgtttgc
     9181 cggatcaaga gctaccaact ctttttccga aggtaactgg cttcagcaga gcgcagatac
     9241 caaatactgt tcttctagtg tagccgtagt taggccacca cttcaagaac tctgtagcac
     9301 cgcctacata cctcgctctg ctaatcctgt taccagtggc tgctgccagt ggcgataagt
     9361 cgtgtcttac cgggttggac tcaagacgat agttaccgga taaggcgcag cggtcgggct
     9421 gaacgggggg ttcgtgcaca cagcccagct tggagcgaac gacctacacc gaactgagat
     9481 acctacagcg tgagctatga gaaagcgcca cgcttcccga agggagaaag gcggacaggt
     9541 atccggtaag cggcagggtc ggaacaggag agcgcacgag ggagcttcca gggggaaacg
     9601 cctggtatct ttatagtcct gtcgggtttc gccacctctg acttgagcgt cgatttttgt
     9661 gatgctcgtc aggggggcgg agcctatgga aaaacgccag caacgcggcc tttttacggt
     9721 tcctggcctt ttgctggcct tttgctcaca tgtgagggcc tatttcccat gattccttca
     9781 tatttgcata tacgatacaa ggctgttaga gagataattg gaattaattt gactgtaaac
     9841 acaaagatat tagtacaaaa tacgtgacgt agaaagtaat aatttcttgg gtagtttgca
     9901 gttttaaaat tatgttttaa aatggactat catatgctta ccgtaacttg aaagtatttc
     9961 gatttcttgg ctttatatat cttgtggaaa ggacgaaaca ccgggtcttc gagaagacct
    10021 gttttagagc tagaaatagc aagttaaaat aaggctagtc cgttatcaac ttgaaaaagt
    10081 ggcaccgagt cggtgctttt ttgttttaga gctagaaata gcaagttaaa ataaggctag
    10141 tccgttttta gcgcgtgcgc caattctgca gacaaatggc tctagaggta ccagtaattc
    10201 atacaaaagg actcgcccct gc
//
