LOCUS       sequence-142288-        9703 bp    DNA     circular SYN 15-DEC-2025
DEFINITION  CRISPR/Cas9 NICKASE plasmid against human Helios (1/2).
ACCESSION   .
VERSION     .
KEYWORDS    .
SOURCE      synthetic DNA construct
  ORGANISM  synthetic DNA construct
REFERENCE   1  (bases 1 to 9703)
  AUTHORS   Gabriel CH, Gross F, Karl M, Stephanowitz H, Hennig AF, Weber M, 
            Gryzik S, Bachmann I, Hecklau K, Wienands J, Schuchhardt J, Herzel 
            H, Radbruch A, Krause E, Baumgrass R
  TITLE     Identification of Novel Nuclear Factor of Activated T Cells 
            (NFAT)-Associated Proteins in T cells.
  JOURNAL   J Biol Chem. 2016 Sep 16. pii: jbc.M116.739326.
  PUBMED    27637333
REFERENCE   2  (bases 1 to 9703)
  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: "J Biol 
            Chem. 2016 Sep 16. pii: jbc.M116.739326."
COMMENT     Sequence Label: Helios-CRISPR-Nick 1/2
            (PX461-EF1a-pSpCas9n(BB...
FEATURES             Location/Qualifiers
     source          1..9703
                     /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        272..347
                     /label=gRNA scaffold
                     /note="guide RNA scaffold for the Streptococcus pyogenes 
                     CRISPR/Cas9 system"
     promoter        460..1638
                     /label=EF-1-alpha promoter
                     /note="strong constitutive promoter for human elongation 
                     factor EF-1-alpha"
     intron          691..1629
                     /label=EF-1-alpha intron A
                     /note="intron upstream of the start codon of human 
                     EF-1-alpha"
     primer_bind     1586..1606
                     /label=EF1a-F
                     /note="Human elongation factor-1a promoter, forward primer"
     regulatory      1659..1668
                     /label=Kozak sequence
                     /note="vertebrate consensus sequence for strong initiation 
                     of translation (Kozak, 1987)"
                     /regulatory_class="other"
     CDS             1668..1733
                     /codon_start=1
                     /product="three tandem FLAG(R) epitope tags, followed by an
                     enterokinase cleavage site"
                     /label=3xFLAG
                     /translation="DYKDHDGDYKDHDIDYKDDDDK"
     CDS             1740..1760
                     /codon_start=1
                     /product="nuclear localization signal of SV40 (simian virus
                     40) large T antigen"
                     /label=SV40 NLS
                     /translation="PKKKRKV"
     CDS             join(1785..1808,1809..1811,1812..5885)
                     /codon_start=1
                     /product="nickase mutant of the Cas9 endonuclease from the 
                     Streptococcus pyogenes Type II CRISPR/Cas system"
                     /label=Cas9(D10A)
                     /note="generates RNA-guided single strand nicks in DNA due 
                     to the D10A mutation in the RuvC catalytic domain"
                     /translation="DKKYSIGLAIGTNSVGWAVITDEYKVPSKKFKVLGNTDRHSIKKN
                     LIGALLFDSGETAEATRLKRTARRRYTRRKNRICYLQEIFSNEMAKVDDSFFHRLEESF
                     LVEEDKKHERHPIFGNIVDEVAYHEKYPTIYHLRKKLVDSTDKADLRLIYLALAHMIKF
                     RGHFLIEGDLNPDNSDVDKLFIQLVQTYNQLFEENPINASGVDAKAILSARLSKSRRLE
                     NLIAQLPGEKKNGLFGNLIALSLGLTPNFKSNFDLAEDAKLQLSKDTYDDDLDNLLAQI
                     GDQYADLFLAAKNLSDAILLSDILRVNTEITKAPLSASMIKRYDEHHQDLTLLKALVRQ
                     QLPEKYKEIFFDQSKNGYAGYIDGGASQEEFYKFIKPILEKMDGTEELLVKLNREDLLR
                     KQRTFDNGSIPHQIHLGELHAILRRQEDFYPFLKDNREKIEKILTFRIPYYVGPLARGN
                     SRFAWMTRKSEETITPWNFEEVVDKGASAQSFIERMTNFDKNLPNEKVLPKHSLLYEYF
                     TVYNELTKVKYVTEGMRKPAFLSGEQKKAIVDLLFKTNRKVTVKQLKEDYFKKIECFDS
                     VEISGVEDRFNASLGTYHDLLKIIKDKDFLDNEENEDILEDIVLTLTLFEDREMIEERL
                     KTYAHLFDDKVMKQLKRRRYTGWGRLSRKLINGIRDKQSGKTILDFLKSDGFANRNFMQ
                     LIHDDSLTFKEDIQKAQVSGQGDSLHEHIANLAGSPAIKKGILQTVKVVDELVKVMGRH
                     KPENIVIEMARENQTTQKGQKNSRERMKRIEEGIKELGSQILKEHPVENTQLQNEKLYL
                     YYLQNGRDMYVDQELDINRLSDYDVDHIVPQSFLKDDSIDNKVLTRSDKNRGKSDNVPS
                     EEVVKKMKNYWRQLLNAKLITQRKFDNLTKAERGGLSELDKAGFIKRQLVETRQITKHV
                     AQILDSRMNTKYDENDKLIREVKVITLKSKLVSDFRKDFQFYKVREINNYHHAHDAYLN
                     AVVGTALIKKYPKLESEFVYGDYKVYDVRKMIAKSEQEIGKATAKYFFYSNIMNFFKTE
                     ITLANGEIRKRPLIETNGETGEIVWDKGRDFATVRKVLSMPQVNIVKKTEVQTGGFSKE
                     SILPKRNSDKLIARKKDWDPKKYGGFDSPTVAYSVLVVAKVEKGKSKKLKSVKELLGIT
                     IMERSSFEKNPIDFLEAKGYKEVKKDLIIKLPKYSLFELENGRKRMLASAGELQKGNEL
                     ALPSKYVNFLYLASHYEKLKGSPEDNEQKQLFVEQHKHYLDEIIEQISEFSKRVILADA
                     NLDKVLSAYNKHRDKPIREQAENIIHLFTLTNLGAPAAFKYFDTTIDRKRYTSTKEVLD
                     ATLIHQSITGLYETRIDLSQLGGD"
     CDS             1785..5885
                     /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="DKKYSIGLAIGTNSVGWAVITDEYKVPSKKFKVLGNTDRHSIKKN
                     LIGALLFDSGETAEATRLKRTARRRYTRRKNRICYLQEIFSNEMAKVDDSFFHRLEESF
                     LVEEDKKHERHPIFGNIVDEVAYHEKYPTIYHLRKKLVDSTDKADLRLIYLALAHMIKF
                     RGHFLIEGDLNPDNSDVDKLFIQLVQTYNQLFEENPINASGVDAKAILSARLSKSRRLE
                     NLIAQLPGEKKNGLFGNLIALSLGLTPNFKSNFDLAEDAKLQLSKDTYDDDLDNLLAQI
                     GDQYADLFLAAKNLSDAILLSDILRVNTEITKAPLSASMIKRYDEHHQDLTLLKALVRQ
                     QLPEKYKEIFFDQSKNGYAGYIDGGASQEEFYKFIKPILEKMDGTEELLVKLNREDLLR
                     KQRTFDNGSIPHQIHLGELHAILRRQEDFYPFLKDNREKIEKILTFRIPYYVGPLARGN
                     SRFAWMTRKSEETITPWNFEEVVDKGASAQSFIERMTNFDKNLPNEKVLPKHSLLYEYF
                     TVYNELTKVKYVTEGMRKPAFLSGEQKKAIVDLLFKTNRKVTVKQLKEDYFKKIECFDS
                     VEISGVEDRFNASLGTYHDLLKIIKDKDFLDNEENEDILEDIVLTLTLFEDREMIEERL
                     KTYAHLFDDKVMKQLKRRRYTGWGRLSRKLINGIRDKQSGKTILDFLKSDGFANRNFMQ
                     LIHDDSLTFKEDIQKAQVSGQGDSLHEHIANLAGSPAIKKGILQTVKVVDELVKVMGRH
                     KPENIVIEMARENQTTQKGQKNSRERMKRIEEGIKELGSQILKEHPVENTQLQNEKLYL
                     YYLQNGRDMYVDQELDINRLSDYDVDHIVPQSFLKDDSIDNKVLTRSDKNRGKSDNVPS
                     EEVVKKMKNYWRQLLNAKLITQRKFDNLTKAERGGLSELDKAGFIKRQLVETRQITKHV
                     AQILDSRMNTKYDENDKLIREVKVITLKSKLVSDFRKDFQFYKVREINNYHHAHDAYLN
                     AVVGTALIKKYPKLESEFVYGDYKVYDVRKMIAKSEQEIGKATAKYFFYSNIMNFFKTE
                     ITLANGEIRKRPLIETNGETGEIVWDKGRDFATVRKVLSMPQVNIVKKTEVQTGGFSKE
                     SILPKRNSDKLIARKKDWDPKKYGGFDSPTVAYSVLVVAKVEKGKSKKLKSVKELLGIT
                     IMERSSFEKNPIDFLEAKGYKEVKKDLIIKLPKYSLFELENGRKRMLASAGELQKGNEL
                     ALPSKYVNFLYLASHYEKLKGSPEDNEQKQLFVEQHKHYLDEIIEQISEFSKRVILADA
                     NLDKVLSAYNKHRDKPIREQAENIIHLFTLTNLGAPAAFKYFDTTIDRKRYTSTKEVLD
                     ATLIHQSITGLYETRIDLSQLGGD"
     CDS             5886..5933
                     /codon_start=1
                     /product="bipartite nuclear localization signal from 
                     nucleoplasmin"
                     /label=nucleoplasmin NLS
                     /translation="KRPAATKKAGQAKKKK"
     CDS             5949..6002
                     /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             join(6003..6005,6006..6716)
                     /codon_start=1
                     /product="enhanced GFP"
                     /label=EGFP
                     /translation="VSKGEELFTGVVPILVELDGDVNGHKFSVSGEGEGDATYGKLTLK
                     FICTTGKLPVPWPTLVTTLTYGVQCFSRYPDHMKQHDFFKSAMPEGYVQERTIFFKDDG
                     NYKTRAEVKFEGDTLVNRIELKGIDFKEDGNILGHKLEYNYNSHNVYIMADKQKNGIKV
                     NFKIRHNIEDGSVQLADHYQQNTPIGDGPVLLPDNHYLSTQSALSKDPNEKRDHMVLLE
                     FVTAAGITLGMDELYK"
     CDS             join(6003..6005,6006..6716)
                     /codon_start=1
                     /product="the original enhanced GFP (Yang et al., 1996)"
                     /label=EGFP
                     /note="mammalian codon-optimized"
                     /translation="VSKGEELFTGVVPILVELDGDVNGHKFSVSGEGEGDATYGKLTLK
                     FICTTGKLPVPWPTLVTTLTYGVQCFSRYPDHMKQHDFFKSAMPEGYVQERTIFFKDDG
                     NYKTRAEVKFEGDTLVNRIELKGIDFKEDGNILGHKLEYNYNSHNVYIMADKQKNGIKV
                     NFKIRHNIEDGSVQLADHYQQNTPIGDGPVLLPDNHYLSTQSALSKDPNEKRDHMVLLE
                     FVTAAGITLGMDELYK"
     primer_bind     complement(6306..6325)
                     /label=EXFP-R
                     /note="For distinguishing EGFP variants, reverse primer"
     primer_bind     6653..6674
                     /label=EGFP-C
                     /note="EGFP, forward primer"
     primer_bind     complement(6744..6761)
                     /label=BGH-rev
                     /note="Bovine growth hormone terminator, reverse primer. 
                     Also called BGH reverse"
     polyA_signal    6750..6957
                     /label=bGH poly(A) signal
                     /note="bovine growth hormone polyadenylation signal"
     repeat_region   6966..7106
                     /label=AAV2 ITR
                     /note="inverted terminal repeat of adeno-associated virus 
                     serotype 2"
     repeat_region   6966..7095
                     /label=AAV2 ITR
     rep_origin      7181..7636
                     /direction=RIGHT
                     /label=f1 ori
                     /note="f1 bacteriophage origin of replication; arrow 
                     indicates direction of (+) strand synthesis"
     primer_bind     complement(7268..7287)
                     /label=F1ori-R
                     /note="F1 origin, reverse primer"
     primer_bind     7478..7499
                     /label=F1ori-F
                     /note="F1 origin, forward primer"
     primer_bind     complement(7653..7672)
                     /label=pRS-marker
                     /note="pRS vectors, use to sequence yeast selectable 
                     marker"
     primer_bind     7772..7794
                     /label=pGEX 3'
                     /note="pGEX vectors, reverse primer"
     primer_bind     complement(7832..7850)
                     /label=pBRforEco
                     /note="pBR322 vectors, upsteam of EcoRI site, forward 
                     primer"
     promoter        7918..8022
                     /gene="bla"
                     /label=AmpR promoter
     CDS             join(8023..8091,8092..8883)
                     /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(8241..8260)
                     /label=Amp-R
                     /note="Ampicillin resistance gene, reverse primer"
     rep_origin      9054..9642
                     /direction=RIGHT
                     /label=ori
                     /note="high-copy-number ColE1/pMB1/pBR322/pUC origin of 
                     replication"
     primer_bind     9543..9562
                     /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 gcatattgga gtgctccctt tgttttagag ctagaaatag caagttaaaa
      301 taaggctagt ccgttatcaa cttgaaaaag tggcaccgag tcggtgcttt tttgttttag
      361 agctagaaat agcaagttaa aataaggcta gtccgttttt agcgcgtgcg ccaattctgc
      421 agacaaatgg ctctagtggt cttgaaagga gtgggaattg gctccggtgc ccgtcagtgg
      481 gcagagcgca catcgcccac agtccccgag aagttggggg gaggggtcgg caattgaacc
      541 ggtgcctaga gaaggtggcg cggggtaaac tgggaaagtg atgtcgtgta ctggctccgc
      601 ctttttcccg agggtggggg agaaccgtat ataagtgcag tagtcgccgt gaacgttctt
      661 tttcgcaacg ggtttgccgc cagaacacag gtaagtgccg tgtgtggttc ccgcgggcct
      721 ggcctcttta cgggttatgg cccttgcgtg ccttgaatta cttccacctg gctgcagtac
      781 gtgattcttg atcccgagct tcgggttgga agtgggtggg agagttcgag gccttgcgct
      841 taaggagccc cttcgcctcg tgcttgagtt gaggcctggc ctgggcgctg gggccgccgc
      901 gtgcgaatct ggtggcacct tcgcgcctgt ctcgctgctt tcgataagtc tctagccatt
      961 taaaattttt gatgacctgc tgcgacgctt tttttctggc aagatagtct tgtaaatgcg
     1021 ggccaagatc tgcacactgg tatttcggtt tttggggccg cgggcggcga cggggcccgt
     1081 gcgtcccagc gcacatgttc ggcgaggcgg ggcctgcgag cgcggccacc gagaatcgga
     1141 cgggggtagt ctcaagctgg ccggcctgct ctggtgcctg gcctcgcgcc gccgtgtatc
     1201 gccccgccct gggcggcaag gctggcccgg tcggcaccag ttgcgtgagc ggaaagatgg
     1261 ccgcttcccg gccctgctgc agggagctca aaatggagga cgcggcgctc gggagagcgg
     1321 gcgggtgagt cacccacaca aaggaaaagg gcctttccgt cctcagccgt cgcttcatgt
     1381 gactccacgg agtaccgggc gccgtccagg cacctcgatt agttctcgag cttttggagt
     1441 acgtcgtctt taggttgggg ggaggggttt tatgcgatgg agtttcccca cactgagtgg
     1501 gtggagactg aagttaggcc agcttggcac ttgatgtaat tctccttgga atttgccctt
     1561 tttgagtttg gatcttggtt cattctcaag cctcagacag tggttcaaag tttttttctt
     1621 ccatttcagg tgtcgtgagg aatttcgaca tttccggtgc caccatggac tataaggacc
     1681 acgacggaga ctacaaggat catgatattg attacaaaga cgatgacgat aagatggccc
     1741 caaagaagaa gcggaaggtc ggtatccacg gagtcccagc agccgacaag aagtacagca
     1801 tcggcctggc catcggcacc aactctgtgg gctgggccgt gatcaccgac gagtacaagg
     1861 tgcccagcaa gaaattcaag gtgctgggca acaccgaccg gcacagcatc aagaagaacc
     1921 tgatcggagc cctgctgttc gacagcggcg aaacagccga ggccacccgg ctgaagagaa
     1981 ccgccagaag aagatacacc agacggaaga accggatctg ctatctgcaa gagatcttca
     2041 gcaacgagat ggccaaggtg gacgacagct tcttccacag actggaagag tccttcctgg
     2101 tggaagagga taagaagcac gagcggcacc ccatcttcgg caacatcgtg gacgaggtgg
     2161 cctaccacga gaagtacccc accatctacc acctgagaaa gaaactggtg gacagcaccg
     2221 acaaggccga cctgcggctg atctatctgg ccctggccca catgatcaag ttccggggcc
     2281 acttcctgat cgagggcgac ctgaaccccg acaacagcga cgtggacaag ctgttcatcc
     2341 agctggtgca gacctacaac cagctgttcg aggaaaaccc catcaacgcc agcggcgtgg
     2401 acgccaaggc catcctgtct gccagactga gcaagagcag acggctggaa aatctgatcg
     2461 cccagctgcc cggcgagaag aagaatggcc tgttcggcaa cctgattgcc ctgagcctgg
     2521 gcctgacccc caacttcaag agcaacttcg acctggccga ggatgccaaa ctgcagctga
     2581 gcaaggacac ctacgacgac gacctggaca acctgctggc ccagatcggc gaccagtacg
     2641 ccgacctgtt tctggccgcc aagaacctgt ccgacgccat cctgctgagc gacatcctga
     2701 gagtgaacac cgagatcacc aaggcccccc tgagcgcctc tatgatcaag agatacgacg
     2761 agcaccacca ggacctgacc ctgctgaaag ctctcgtgcg gcagcagctg cctgagaagt
     2821 acaaagagat tttcttcgac cagagcaaga acggctacgc cggctacatt gacggcggag
     2881 ccagccagga agagttctac aagttcatca agcccatcct ggaaaagatg gacggcaccg
     2941 aggaactgct cgtgaagctg aacagagagg acctgctgcg gaagcagcgg accttcgaca
     3001 acggcagcat cccccaccag atccacctgg gagagctgca cgccattctg cggcggcagg
     3061 aagattttta cccattcctg aaggacaacc gggaaaagat cgagaagatc ctgaccttcc
     3121 gcatccccta ctacgtgggc cctctggcca ggggaaacag cagattcgcc tggatgacca
     3181 gaaagagcga ggaaaccatc accccctgga acttcgagga agtggtggac aagggcgctt
     3241 ccgcccagag cttcatcgag cggatgacca acttcgataa gaacctgccc aacgagaagg
     3301 tgctgcccaa gcacagcctg ctgtacgagt acttcaccgt gtataacgag ctgaccaaag
     3361 tgaaatacgt gaccgaggga atgagaaagc ccgccttcct gagcggcgag cagaaaaagg
     3421 ccatcgtgga cctgctgttc aagaccaacc ggaaagtgac cgtgaagcag ctgaaagagg
     3481 actacttcaa gaaaatcgag tgcttcgact ccgtggaaat ctccggcgtg gaagatcggt
     3541 tcaacgcctc cctgggcaca taccacgatc tgctgaaaat tatcaaggac aaggacttcc
     3601 tggacaatga ggaaaacgag gacattctgg aagatatcgt gctgaccctg acactgtttg
     3661 aggacagaga gatgatcgag gaacggctga aaacctatgc ccacctgttc gacgacaaag
     3721 tgatgaagca gctgaagcgg cggagataca ccggctgggg caggctgagc cggaagctga
     3781 tcaacggcat ccgggacaag cagtccggca agacaatcct ggatttcctg aagtccgacg
     3841 gcttcgccaa cagaaacttc atgcagctga tccacgacga cagcctgacc tttaaagagg
     3901 acatccagaa agcccaggtg tccggccagg gcgatagcct gcacgagcac attgccaatc
     3961 tggccggcag ccccgccatt aagaagggca tcctgcagac agtgaaggtg gtggacgagc
     4021 tcgtgaaagt gatgggccgg cacaagcccg agaacatcgt gatcgaaatg gccagagaga
     4081 accagaccac ccagaaggga cagaagaaca gccgcgagag aatgaagcgg atcgaagagg
     4141 gcatcaaaga gctgggcagc cagatcctga aagaacaccc cgtggaaaac acccagctgc
     4201 agaacgagaa gctgtacctg tactacctgc agaatgggcg ggatatgtac gtggaccagg
     4261 aactggacat caaccggctg tccgactacg atgtggacca tatcgtgcct cagagctttc
     4321 tgaaggacga ctccatcgac aacaaggtgc tgaccagaag cgacaagaac cggggcaaga
     4381 gcgacaacgt gccctccgaa gaggtcgtga agaagatgaa gaactactgg cggcagctgc
     4441 tgaacgccaa gctgattacc cagagaaagt tcgacaatct gaccaaggcc gagagaggcg
     4501 gcctgagcga actggataag gccggcttca tcaagagaca gctggtggaa acccggcaga
     4561 tcacaaagca cgtggcacag atcctggact cccggatgaa cactaagtac gacgagaatg
     4621 acaagctgat ccgggaagtg aaagtgatca ccctgaagtc caagctggtg tccgatttcc
     4681 ggaaggattt ccagttttac aaagtgcgcg agatcaacaa ctaccaccac gcccacgacg
     4741 cctacctgaa cgccgtcgtg ggaaccgccc tgatcaaaaa gtaccctaag ctggaaagcg
     4801 agttcgtgta cggcgactac aaggtgtacg acgtgcggaa gatgatcgcc aagagcgagc
     4861 aggaaatcgg caaggctacc gccaagtact tcttctacag caacatcatg aactttttca
     4921 agaccgagat taccctggcc aacggcgaga tccggaagcg gcctctgatc gagacaaacg
     4981 gcgaaaccgg ggagatcgtg tgggataagg gccgggattt tgccaccgtg cggaaagtgc
     5041 tgagcatgcc ccaagtgaat atcgtgaaaa agaccgaggt gcagacaggc ggcttcagca
     5101 aagagtctat cctgcccaag aggaacagcg ataagctgat cgccagaaag aaggactggg
     5161 accctaagaa gtacggcggc ttcgacagcc ccaccgtggc ctattctgtg ctggtggtgg
     5221 ccaaagtgga aaagggcaag tccaagaaac tgaagagtgt gaaagagctg ctggggatca
     5281 ccatcatgga aagaagcagc ttcgagaaga atcccatcga ctttctggaa gccaagggct
     5341 acaaagaagt gaaaaaggac ctgatcatca agctgcctaa gtactccctg ttcgagctgg
     5401 aaaacggccg gaagagaatg ctggcctctg ccggcgaact gcagaaggga aacgaactgg
     5461 ccctgccctc caaatatgtg aacttcctgt acctggccag ccactatgag aagctgaagg
     5521 gctcccccga ggataatgag cagaaacagc tgtttgtgga acagcacaag cactacctgg
     5581 acgagatcat cgagcagatc agcgagttct ccaagagagt gatcctggcc gacgctaatc
     5641 tggacaaagt gctgtccgcc tacaacaagc accgggataa gcccatcaga gagcaggccg
     5701 agaatatcat ccacctgttt accctgacca atctgggagc ccctgccgcc ttcaagtact
     5761 ttgacaccac catcgaccgg aagaggtaca ccagcaccaa agaggtgctg gacgccaccc
     5821 tgatccacca gagcatcacc ggcctgtacg agacacggat cgacctgtct cagctgggag
     5881 gcgacaaaag gccggcggcc acgaaaaagg ccggccaggc aaaaaagaaa aaggaattcg
     5941 gcagtggaga gggcagagga agtctgctaa catgcggtga cgtcgaggag aatcctggcc
     6001 cagtgagcaa gggcgaggag ctgttcaccg gggtggtgcc catcctggtc gagctggacg
     6061 gcgacgtaaa cggccacaag ttcagcgtgt ccggcgaggg cgagggcgat gccacctacg
     6121 gcaagctgac cctgaagttc atctgcacca ccggcaagct gcccgtgccc tggcccaccc
     6181 tcgtgaccac cctgacctac ggcgtgcagt gcttcagccg ctaccccgac cacatgaagc
     6241 agcacgactt cttcaagtcc gccatgcccg aaggctacgt ccaggagcgc accatcttct
     6301 tcaaggacga cggcaactac aagacccgcg ccgaggtgaa gttcgagggc gacaccctgg
     6361 tgaaccgcat cgagctgaag ggcatcgact tcaaggagga cggcaacatc ctggggcaca
     6421 agctggagta caactacaac agccacaacg tctatatcat ggccgacaag cagaagaacg
     6481 gcatcaaggt gaacttcaag atccgccaca acatcgagga cggcagcgtg cagctcgccg
     6541 accactacca gcagaacacc cccatcggcg acggccccgt gctgctgccc gacaaccact
     6601 acctgagcac ccagtccgcc ctgagcaaag accccaacga gaagcgcgat cacatggtcc
     6661 tgctggagtt cgtgaccgcc gccgggatca ctctcggcat ggacgagctg tacaaggaat
     6721 tctaactaga gctcgctgat cagcctcgac tgtgccttct agttgccagc catctgttgt
     6781 ttgcccctcc cccgtgcctt ccttgaccct ggaaggtgcc actcccactg tcctttccta
     6841 ataaaatgag gaaattgcat cgcattgtct gagtaggtgt cattctattc tggggggtgg
     6901 ggtggggcag gacagcaagg gggaggattg ggaagagaat agcaggcatg ctggggagcg
     6961 gccgcaggaa cccctagtga tggagttggc cactccctct ctgcgcgctc gctcgctcac
     7021 tgaggccggg cgaccaaagg tcgcccgacg cccgggcttt gcccgggcgg cctcagtgag
     7081 cgagcgagcg cgcagctgcc tgcaggggcg cctgatgcgg tattttctcc ttacgcatct
     7141 gtgcggtatt tcacaccgca tacgtcaaag caaccatagt acgcgccctg tagcggcgca
     7201 ttaagcgcgg cgggtgtggt ggttacgcgc agcgtgaccg ctacacttgc cagcgcccta
     7261 gcgcccgctc ctttcgcttt cttcccttcc tttctcgcca cgttcgccgg ctttccccgt
     7321 caagctctaa atcgggggct ccctttaggg ttccgattta gtgctttacg gcacctcgac
     7381 cccaaaaaac ttgatttggg tgatggttca cgtagtgggc catcgccctg atagacggtt
     7441 tttcgccctt tgacgttgga gtccacgttc tttaatagtg gactcttgtt ccaaactgga
     7501 acaacactca accctatctc gggctattct tttgatttat aagggatttt gccgatttcg
     7561 gcctattggt taaaaaatga gctgatttaa caaaaattta acgcgaattt taacaaaata
     7621 ttaacgttta caattttatg gtgcactctc agtacaatct gctctgatgc cgcatagtta
     7681 agccagcccc gacacccgcc aacacccgct gacgcgccct gacgggcttg tctgctcccg
     7741 gcatccgctt acagacaagc tgtgaccgtc tccgggagct gcatgtgtca gaggttttca
     7801 ccgtcatcac cgaaacgcgc gagacgaaag ggcctcgtga tacgcctatt tttataggtt
     7861 aatgtcatga taataatggt ttcttagacg tcaggtggca cttttcgggg aaatgtgcgc
     7921 ggaaccccta tttgtttatt tttctaaata cattcaaata tgtatccgct catgagacaa
     7981 taaccctgat aaatgcttca ataatattga aaaaggaaga gtatgagtat tcaacatttc
     8041 cgtgtcgccc ttattccctt ttttgcggca ttttgccttc ctgtttttgc tcacccagaa
     8101 acgctggtga aagtaaaaga tgctgaagat cagttgggtg cacgagtggg ttacatcgaa
     8161 ctggatctca acagcggtaa gatccttgag agttttcgcc ccgaagaacg ttttccaatg
     8221 atgagcactt ttaaagttct gctatgtggc gcggtattat cccgtattga cgccgggcaa
     8281 gagcaactcg gtcgccgcat acactattct cagaatgact tggttgagta ctcaccagtc
     8341 acagaaaagc atcttacgga tggcatgaca gtaagagaat tatgcagtgc tgccataacc
     8401 atgagtgata acactgcggc caacttactt ctgacaacga tcggaggacc gaaggagcta
     8461 accgcttttt tgcacaacat gggggatcat gtaactcgcc ttgatcgttg ggaaccggag
     8521 ctgaatgaag ccataccaaa cgacgagcgt gacaccacga tgcctgtagc aatggcaaca
     8581 acgttgcgca aactattaac tggcgaacta cttactctag cttcccggca acaattaata
     8641 gactggatgg aggcggataa agttgcagga ccacttctgc gctcggccct tccggctggc
     8701 tggtttattg ctgataaatc tggagccggt gagcgtggaa gccgcggtat cattgcagca
     8761 ctggggccag atggtaagcc ctcccgtatc gtagttatct acacgacggg gagtcaggca
     8821 actatggatg aacgaaatag acagatcgct gagataggtg cctcactgat taagcattgg
     8881 taactgtcag accaagttta ctcatatata ctttagattg atttaaaact tcatttttaa
     8941 tttaaaagga tctaggtgaa gatccttttt gataatctca tgaccaaaat cccttaacgt
     9001 gagttttcgt tccactgagc gtcagacccc gtagaaaaga tcaaaggatc ttcttgagat
     9061 cctttttttc tgcgcgtaat ctgctgcttg caaacaaaaa aaccaccgct accagcggtg
     9121 gtttgtttgc cggatcaaga gctaccaact ctttttccga aggtaactgg cttcagcaga
     9181 gcgcagatac caaatactgt ccttctagtg tagccgtagt taggccacca cttcaagaac
     9241 tctgtagcac cgcctacata cctcgctctg ctaatcctgt taccagtggc tgctgccagt
     9301 ggcgataagt cgtgtcttac cgggttggac tcaagacgat agttaccgga taaggcgcag
     9361 cggtcgggct gaacgggggg ttcgtgcaca cagcccagct tggagcgaac gacctacacc
     9421 gaactgagat acctacagcg tgagctatga gaaagcgcca cgcttcccga agggagaaag
     9481 gcggacaggt atccggtaag cggcagggtc ggaacaggag agcgcacgag ggagcttcca
     9541 gggggaaacg cctggtatct ttatagtcct gtcgggtttc gccacctctg acttgagcgt
     9601 cgatttttgt gatgctcgtc aggggggcgg agcctatgga aaaacgccag caacgcggcc
     9661 tttttacggt tcctggcctt ttgctggcct tttgctcaca tgt
//
