LOCUS       sequence-208338-        9823 bp    DNA     circular SYN 15-DEC-2025
DEFINITION  The designed sgRNA cloned into this plasmid directs the specific DNA
            cleavage exerted by Cas9 nuclease in a region of exon 5 of the human
            TK2 gene. The plasmid also includes the Cas9 nuclease and CD4..
ACCESSION   .
VERSION     .
KEYWORDS    .
SOURCE      synthetic DNA construct
  ORGANISM  synthetic DNA construct
REFERENCE   1  (bases 1 to 9823)
  AUTHORS   Meca-Cortes O, Guerra-Rebollo M, Garrido C, Borros S, Rubio N, 
            Blanco J
  TITLE     CRISPR/Cas9-Mediated Knockin Application in Cell Therapy: A 
            Non-viral Procedure for Bystander Treatment of Glioma in Mice.
  JOURNAL   Mol Ther Nucleic Acids. 2017 Sep 15;8:395-403. doi: 
            10.1016/j.omtn.2017.07.012. Epub 2017 Jul 18.
  PUBMED    28918039
REFERENCE   2  (bases 1 to 9823)
  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: "Mol Ther 
            Nucleic Acids."; date: "2017-09-15"; volume: "8:395-403. doi"; 
            pages: " 10.1016/j.omtn.2017.07.012. Epub 2017 Jul 18"
COMMENT     Sequence Label: pCRISPR-Cas9/CD4-TK2
FEATURES             Location/Qualifiers
     source          1..9823
                     /mol_type="other DNA"
                     /organism="synthetic DNA construct"
     primer_bind     complement(46..65)
                     /label=pRS-marker
                     /note="pRS vectors, use to sequence yeast selectable 
                     marker"
     enhancer        237..616
                     /label=CMV enhancer
                     /note="human cytomegalovirus immediate early enhancer"
     promoter        617..820
                     /label=CMV promoter
                     /note="human cytomegalovirus (CMV) immediate early 
                     promoter"
     primer_bind     771..791
                     /label=CMV-F
                     /note="Human CMV immediate early promoter, forward primer"
     primer_bind     817..841
                     /label=LNCX
                     /note="Human CMV promoter, forward primer"
     regulatory      915..924
                     /label=Kozak sequence
                     /note="vertebrate consensus sequence for strong initiation 
                     of translation (Kozak, 1987)"
                     /regulatory_class="other"
     CDS             924..965
                     /codon_start=1
                     /product="epitope tag from simian virus 5"
                     /label=V5 tag
                     /translation="GKPIPNPLLGLDST"
     CDS             969..989
                     /codon_start=1
                     /product="nuclear localization signal of SV40 (simian virus
                     40) large T antigen"
                     /label=SV40 NLS
                     /translation="PKKKRKV"
     CDS             1014..5114
                     /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             1014..5114
                     /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"
     RBS             5027..5035
                     /label=Shine-Dalgarno sequence
                     /note="full consensus sequence for ribosome-binding sites 
                     upstream of start codons in E. coli; complementary to a 
                     region in the 3' end of the 16S rRNA (Chen et al., 1994)"
     CDS             5127..5147
                     /codon_start=1
                     /product="nuclear localization signal of SV40 (simian virus
                     40) large T antigen"
                     /label=SV40 NLS
                     /translation="PKKKRKV"
     CDS             5169..5222
                     /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(5223..5297,5298..6494)
                     /codon_start=1
                     /product="truncated human CD4 cell surface glycoprotein 
                     lacking the cytoplasmic tail"
                     /label=CD4 (truncated)
                     /translation="MNRGVPFRHLLLVLQLALLPAATQGKKVVLGKKGDTVELTCTASQ
                     KKSIQFHWKNSNQIKILGNQGSFLTKGPSKLNDRADSRRSLWDQGNFPLIIKNLKIEDS
                     DTYICEVEDQKEEVQLLVFGLTANSDTHLLQGQSLTLTLESPPGSSPSVQCRSPRGKNI
                     QGGKTLSVSQLELQDSGTWTCTVLQNQKKVEFKIDIVVLAFQKASSIVYKKEGEQVEFS
                     FPLAFTVEKLTGSGELWWQAERASSSKSWITFDLKNKEVSVKRVTQDPKLQMGKKLPLH
                     LTLPQALPQYAGSGNLTLALEAKTGKLHQEVNLVVMRATQLQKNLTCEVWGPTSPKLML
                     SLKLENKEAKVSKREKAVWVLNPEAGMWQCLLSDSGQVLLESNIKVLPTWSTPVQPMAL
                     IVLGGVAGLLLFIGLGIFFCVRCRH"
     primer_bind     complement(6531..6550)
                     /label=TK-pA-R
                     /note="Thymidine kinase polyA, reverse primer"
     polyA_signal    6575..6623
                     /label=HSV TK poly(A) signal
                     /note="herpes simplex virus thymidine kinase 
                     polyadenylation signal (Cole and Stacy, 1985)"
     rep_origin      6825..7253
                     /direction=RIGHT
                     /label=f1 ori
                     /note="f1 bacteriophage origin of replication; arrow 
                     indicates direction of (+) strand synthesis"
     primer_bind     complement(6912..6931)
                     /label=F1ori-R
                     /note="F1 origin, reverse primer"
     primer_bind     7122..7143
                     /label=F1ori-F
                     /note="F1 origin, forward primer"
     promoter        7273..7521
                     /label=U6 promoter
                     /note="RNA polymerase III promoter for human U6 snRNA 
                     (Domitrovich & Kunkel, 2003)"
     primer_bind     7273..7293
                     /label=hU6-F
                     /note="Human U6 promoter, forward primer"
     primer_bind     7444..7463
                     /label=LKO.1 5'
                     /note="Human U6 promoter, forward primer"
     misc_RNA        7543..7618
                     /label=gRNA scaffold
                     /note="guide RNA scaffold for the Streptococcus pyogenes 
                     CRISPR/Cas9 system"
     primer_bind     complement(7668..7684)
                     /label=M13 rev
                     /note="common sequencing primer, one of multiple similar 
                     variants"
     primer_bind     complement(7668..7684)
                     /label=M13 Reverse
                     /note="In lacZ gene. Also called M13-rev"
     primer_bind     complement(7681..7703)
                     /label=M13/pUC Reverse
                     /note="In lacZ gene"
     protein_bind    7692..7708
                     /label=lac operator
                     /bound_moiety="lac repressor encoded by lacI"
                     /note="The lac repressor binds to the lac operator to 
                     inhibit transcription in E. coli. This inhibition can be 
                     relieved by adding lactose or 
                     isopropyl-beta-D-thiogalactopyranoside (IPTG)."
     promoter        complement(join(7716..7722,7723..7740,7741..7746))
                     /label=lac promoter
                     /note="promoter for the E. coli lac operon"
     protein_bind    7761..7782
                     /label=CAP binding site
                     /bound_moiety="E. coli catabolite activator protein"
                     /note="CAP binding activates transcription in the presence 
                     of cAMP."
     primer_bind     complement(7899..7916)
                     /label=L4440
                     /note="L4440 vector, forward primer"
     rep_origin      complement(8070..8658)
                     /direction=LEFT
                     /label=ori
                     /note="high-copy-number ColE1/pMB1/pBR322/pUC origin of 
                     replication"
     primer_bind     complement(8150..8169)
                     /label=pBR322ori-F
                     /note="pBR322 origin, forward primer"
     CDS             complement(join(8829..9620,9621..9689))
                     /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     9452..9471
                     /label=Amp-R
                     /note="Ampicillin resistance gene, reverse primer"
     promoter        complement(9690..9794)
                     /gene="bla"
                     /label=AmpR promoter
ORIGIN
        1 tcgacggatc gggagatctc ccgatcccct atggtgcact ctcagtacaa tctgctctga
       61 tgccgcatag ttaagccagt atctgctccc tgcttgtgtg ttggaggtcg ctgagtagtg
      121 cgcgagcaaa atttaagcta caacaaggca aggcttgacc gacaattgca tgaagaatct
      181 gcttagggtt aggcgttttg cgctgcttcg cgatgtacgg gccagatata cgcgttgaca
      241 ttgattattg actagttatt aatagtaatc aattacgggg tcattagttc atagcccata
      301 tatggagttc cgcgttacat aacttacggt aaatggcccg cctggctgac cgcccaacga
      361 cccccgccca ttgacgtcaa taatgacgta tgttcccata gtaacgccaa tagggacttt
      421 ccattgacgt caatgggtgg agtatttacg gtaaactgcc cacttggcag tacatcaagt
      481 gtatcatatg ccaagtacgc cccctattga cgtcaatgac ggtaaatggc ccgcctggca
      541 ttatgcccag tacatgacct tatgggactt tcctacttgg cagtacatct acgtattagt
      601 catcgctatt accatggtga tgcggttttg gcagtacatc aatgggcgtg gatagcggtt
      661 tgactcacgg ggatttccaa gtctccaccc cattgacgtc aatgggagtt tgttttggca
      721 ccaaaatcaa cgggactttc caaaatgtcg taacaactcc gccccattga cgcaaatggg
      781 cggtaggcgt gtacggtggg aggtctatat aagcagagct cgtttagtga accgtcagat
      841 cgcctggaga cgccatccac gctgttttga cctccataga agacaccggg accgatccag
      901 cctccggagc ggccgccacc atgggcaagc ccatccctaa ccccctgttg gggctggaca
      961 gcaccgctcc caaaaagaaa aggaaggtgg gcattcacgg cgtgcctgcg gccgacaaaa
     1021 agtacagcat cggccttgat atcggcacca atagcgtggg ctgggccgtt atcacagacg
     1081 aatacaaggt acccagcaag aagttcaagg tgctggggaa tacagacagg cactctatca
     1141 agaaaaacct tatcggggct ctgctgtttg actcaggcga gaccgccgag gccaccaggt
     1201 tgaagaggac cgcaaggcga aggtacaccc ggaggaagaa caggatctgc tatctgcagg
     1261 agatcttcag caacgagatg gccaaggtgg acgacagctt cttccacagg ctggaggaga
     1321 gcttccttgt cgaggaggat aagaagcacg aacgacaccc catcttcggc aacatagtcg
     1381 acgaggtcgc ttatcacgag aagtacccca ccatctacca cctgcgaaag aaattggtgg
     1441 atagcaccga taaagccgac ttgcgactta tctacttggc tctggcgcac atgattaagt
     1501 tcaggggcca cttcctgatc gagggcgacc ttaaccccga caacagtgac gtagacaaat
     1561 tgttcatcca gcttgtacag acctataacc agctgttcga ggaaaaccct attaacgcca
     1621 gcggggtgga tgcgaaggcc atacttagcg ccaggctgag caaaagcagg cgcttggaga
     1681 acctgatagc ccagctgccc ggtgaaaaga agaacggcct cttcggtaat ctgattgccc
     1741 tgagcctggg cctgaccccc aacttcaaga gcaacttcga cctggcagaa gatgccaagc
     1801 tgcagttgag taaggacacc tatgacgacg acttggacaa tctgctcgcc caaatcggcg
     1861 accagtacgc tgacctgttc ctcgccgcca agaacctttc tgacgcaatc ctgcttagcg
     1921 atatccttag ggtgaacaca gagatcacca aggcccccct gagcgccagc atgatcaaga
     1981 ggtacgacga gcaccatcag gacctgaccc ttctgaaggc cctggtgagg cagcaactgc
     2041 ccgagaagta caaggagatc tttttcgacc agagcaagaa cggctacgcc ggctacatcg
     2101 acggcggagc cagccaagag gagttctaca agttcatcaa gcccatcctg gagaagatgg
     2161 atggcaccga ggagctgctg gtgaagctga acagggaaga tttgctccgg aagcagagga
     2221 cctttgacaa cggtagcatc ccccaccaga tccacctggg cgagctgcac gcaatactga
     2281 ggcgacagga ggatttctac cccttcctca aggacaatag ggagaaaatc gaaaagattc
     2341 tgaccttcag gatcccctac tacgtgggcc ctcttgccag gggcaacagc cgattcgctt
     2401 ggatgacaag aaagagcgag gagaccatca ccccctggaa cttcgaggaa gtggtggaca
     2461 aaggagcaag cgcgcagtct ttcatcgaac ggatgaccaa tttcgacaaa aacctgccta
     2521 acgagaaggt gctgcccaag cacagcctgc tttacgagta cttcaccgtg tacaacgagc
     2581 tcaccaaggt gaaatatgtg accgagggca tgcgaaaacc cgctttcctg agcggcgagc
     2641 agaagaaggc catcgtggac ctgctgttca agaccaacag gaaggtgacc gtgaagcagc
     2701 tgaaggagga ctacttcaag aagatcgagt gctttgatag cgtggaaata agcggcgtgg
     2761 aggacaggtt caacgccagc ctgggcacct accacgactt gttgaagata atcaaagaca
     2821 aggatttcct ggataatgag gagaacgagg atatactcga ggacatcgtg ctgactttga
     2881 ccctgtttga ggaccgagag atgattgaag aaaggctcaa aacctacgcc cacctgttcg
     2941 acgacaaagt gatgaaacaa ctgaagagac gaagatacac cggctggggc agactgtcca
     3001 ggaagctcat caacggcatt agggacaagc agagcggcaa gaccatcctg gatttcctga
     3061 agtccgacgg cttcgccaac cgaaacttca tgcagctgat tcacgatgac agcttgacct
     3121 tcaaggagga catccagaag gcccaggtta gcggccaggg cgactccctg cacgaacata
     3181 ttgcaaacct ggcaggctcc cctgcgatca agaagggcat actgcagacc gttaaggttg
     3241 tggacgaatt ggtcaaggtc atgggcaggc acaagcccga aaacatagtt atagagatgg
     3301 ccagagagaa ccagaccacc caaaagggcc agaagaacag ccgggagcgc atgaaaagga
     3361 tcgaggaggg tatcaaggaa ctcggaagcc agatcctcaa agagcacccc gtggagaata
     3421 cccagctcca gaacgagaag ctgtacctgt actacctgca gaacggcagg gacatgtacg
     3481 ttgaccagga gttggacatc aacaggcttt cagactatga cgtggatcac atagtgcccc
     3541 agagctttct taaagacgat agcatcgaca acaaggtcct gacccgctcc gacaaaaaca
     3601 ggggcaaaag cgacaacgtg ccaagcgaag aggtggttaa aaagatgaag aactactgga
     3661 ggcaactgct caacgcgaaa ttgatcaccc agagaaagtt cgataacctg accaaggccg
     3721 agaggggcgg actctccgaa cttgacaaag cgggcttcat aaagaggcag ctggtcgaga
     3781 cccgacagat cacgaagcac gtggcccaaa tcctcgacag cagaatgaat accaagtacg
     3841 atgagaatga caaactcatc agggaagtga aagtgattac cctgaagagc aagttggtgt
     3901 ccgactttcg caaagatttc cagttctaca aggtgaggga gatcaacaac taccaccatg
     3961 cccacgacgc atacctgaac gccgtggtcg gcaccgccct gattaagaag tatccaaagc
     4021 tggagtccga atttgtctac ggcgactaca aagtttacga tgtgaggaag atgatcgcta
     4081 agagcgaaca ggagatcggc aaggccaccg ctaagtattt cttctacagc aacatcatga
     4141 actttttcaa gaccgagatc acacttgcca acggcgaaat caggaagagg ccgcttatcg
     4201 agaccaacgg tgagaccggc gagatcgtgt gggacaaggg cagggacttc gccaccgtga
     4261 ggaaagtcct gagcatgccc caggtgaata ttgtgaaaaa aactgaggtg cagacaggcg
     4321 gctttagcaa ggaatccatc ctgcccaaga ggaacagcga caagctgatc gcccggaaga
     4381 aggactggga ccctaagaag tatggaggct tcgacagccc caccgtagcc tacagcgtgc
     4441 tggtggtcgc gaaggtagag aaggggaaga gcaagaaact gaagagcgtg aaggagctgc
     4501 tcggcataac catcatggag aggtccagct ttgagaagaa ccccattgac tttttggaag
     4561 ccaagggcta caaagaggtc aaaaaggacc tgatcatcaa actccccaag tactccctgt
     4621 ttgaattgga gaacggcaga aagaggatgc tggcgagcgc tggggaactg caaaagggca
     4681 acgaactggc gctgcccagc aagtacgtga attttctgta cctggcgtcc cactacgaaa
     4741 agctgaaagg cagccccgag gacaacgagc agaagcagct gttcgtggag cagcacaagc
     4801 attacctgga cgagataatc gagcaaatca gcgagttcag caagagggtg attctggccg
     4861 acgcgaacct ggataaggtc ctcagcgcct acaacaagca ccgagacaaa cccatcaggg
     4921 agcaggccga gaatatcata cacctgttca ccctgacaaa tctgggcgca cctgcggcat
     4981 tcaaatactt cgataccacc atcgacagga aaaggtacac tagcactaag gaggtgctgg
     5041 atgccacctt gatccaccag tccattaccg gcctgtatga gaccaggatc gacctgagcc
     5101 agcttggagg cgactctagg gcggacccaa aaaagaaaag gaaggtggaa ttctctagag
     5161 gcagtggaga gggcagagga agtctgctaa catgcggtga cgtcgaggag aatcctggcc
     5221 caatgaaccg gggagtccct tttaggcact tgcttctggt gctgcaactg gcgctcctcc
     5281 cagcagccac tcagggaaag aaagtggtgc tgggcaaaaa aggggataca gtggaactga
     5341 cctgtacagc ttcccagaag aagagcatac aattccactg gaaaaactcc aaccagataa
     5401 agattctggg aaatcagggc tccttcttaa ctaaaggtcc atccaagctg aatgatcgcg
     5461 ctgactcaag aagaagcctt tgggaccaag gaaacttccc cctgatcatc aagaatctta
     5521 agatagaaga ctcagatact tacatctgtg aagtggagga ccagaaggag gaggtgcaat
     5581 tgctagtgtt cggattgact gccaactctg acacccacct gcttcagggg cagagcctga
     5641 ccctgacctt ggagagcccc cctggtagta gcccctcagt gcaatgtagg agtccaaggg
     5701 gtaaaaacat acaggggggg aagaccctct ccgtgtctca gctggagctc caggatagtg
     5761 gcacctggac atgcactgtc ttgcagaacc agaagaaggt ggagttcaaa atagacatcg
     5821 tggtgctagc tttccagaag gcctccagca tagtctataa gaaagagggg gaacaggtgg
     5881 agttctcctt cccactcgcc tttacagttg aaaagctgac gggcagtggc gagctgtggt
     5941 ggcaggcgga gagggcttcc tcctccaagt cttggatcac ctttgacctg aagaacaagg
     6001 aagtgtctgt aaaacgggtt acccaggacc ctaagctcca gatgggcaag aagctcccgc
     6061 tccacctcac cctgccccag gccttgcctc agtatgctgg ctctggaaac ctcaccctgg
     6121 cccttgaagc gaaaacagga aagttgcatc aggaagtgaa cctggtggtg atgagagcca
     6181 ctcagctcca gaaaaatttg acctgtgagg tgtggggacc cacctcccct aagctgatgc
     6241 tgagcttgaa actggagaac aaggaggcaa aggtctcgaa gcgggagaag gcggtgtggg
     6301 tgctgaaccc tgaggcgggg atgtggcagt gtctgctgag tgactcggga caggtcctgc
     6361 tggaatccaa catcaaggtt ctgcccacat ggtcgacccc ggtgcagcca atggccctga
     6421 ttgtgctggg gggcgtcgcc ggcctcctgc ttttcattgg gctaggcatc ttcttctgtg
     6481 tcaggtgccg gcacaccggt tagtaatgag tttaaacggg ggaggctaac tgaaacacgg
     6541 aaggagacaa taccggaagg aacccgcgct atgacggcaa taaaaagaca gaataaaacg
     6601 cacgggtgtt gggtcgtttg ttcataaacg cggggttcgg tcccagggct ggcactctgt
     6661 cgatacccca ccgagacccc attggggcca atacgcccgc gtttcttcct tttccccacc
     6721 ccacccccca agttcgggtg aaggcccagg gctcgcagcc aacgtcgggg cggcaggccc
     6781 tgccatagca gatctgcgca gctggggctc tagggggtat ccccacgcgc cctgtagcgg
     6841 cgcattaagc gcggcgggtg tggtggttac gcgcagcgtg accgctacac ttgccagcgc
     6901 cctagcgccc gctcctttcg ctttcttccc ttcctttctc gccacgttcg ccggctttcc
     6961 ccgtcaagct ctaaatcggg ggctcccttt agggttccga tttagtgctt tacggcacct
     7021 cgaccccaaa aaacttgatt agggtgatgg ttcacgtagt gggccatcgc cctgatagac
     7081 ggtttttcgc cctttgacgt tggagtccac gttctttaat agtggactct tgttccaaac
     7141 tggaacaaca ctcaacccta tctcggtcta ttcttttgat ttataaggga ttttgccgat
     7201 ttcggcctat tggttaaaaa atgagctgat ttaacaaaaa tttaacgcga attaattaag
     7261 gtcgggcagg aagagggcct atttcccatg attccttcat atttgcatat acgatacaag
     7321 gctgttagag agataattag aattaatttg actgtaaaca caaagatatt agtacaaaat
     7381 acgtgacgta gaaagtaata atttcttggg tagtttgcag ttttaaaatt atgttttaaa
     7441 atggactatc atatgcttac cgtaacttga aagtatttcg atttcttggc tttatatatc
     7501 ttgtggaaag gacgaaacac cgtgtaccac gatgcctctc gcgttttaga gctagaaata
     7561 gcaagttaaa ataaggctag tccgttatca acttgaaaaa gtggcaccga gtcggtgctt
     7621 ttttctagta taccgtcgac ctctagctag agcttggcgt aatcatggtc atagctgttt
     7681 cctgtgtgaa attgttatcc gctcacaatt ccacacaaca tacgagccgg aagcataaag
     7741 tgtaaagcct ggggtgccta atgagtgagc taactcacat taattgcgtt gcgctcactg
     7801 cccgctttcc agtcgggaaa cctgtcgtgc cagctgcatt aatgaatcgg ccaacgcgcg
     7861 gggagaggcg gtttgcgtat tgggcgctct tccgcttcct cgctcactga ctcgctgcgc
     7921 tcggtcgttc ggctgcggcg agcggtatca gctcactcaa aggcggtaat acggttatcc
     7981 acagaatcag gggataacgc aggaaagaac atgtgagcaa aaggccagca aaaggccagg
     8041 aaccgtaaaa aggccgcgtt gctggcgttt ttccataggc tccgcccccc tgacgagcat
     8101 cacaaaaatc gacgctcaag tcagaggtgg cgaaacccga caggactata aagataccag
     8161 gcgtttcccc ctggaagctc cctcgtgcgc tctcctgttc cgaccctgcc gcttaccgga
     8221 tacctgtccg cctttctccc ttcgggaagc gtggcgcttt ctcatagctc acgctgtagg
     8281 tatctcagtt cggtgtaggt cgttcgctcc aagctgggct gtgtgcacga accccccgtt
     8341 cagcccgacc gctgcgcctt atccggtaac tatcgtcttg agtccaaccc ggtaagacac
     8401 gacttatcgc cactggcagc agccactggt aacaggatta gcagagcgag gtatgtaggc
     8461 ggtgctacag agttcttgaa gtggtggcct aactacggct acactagaag aacagtattt
     8521 ggtatctgcg ctctgctgaa gccagttacc ttcggaaaaa gagttggtag ctcttgatcc
     8581 ggcaaacaaa ccaccgctgg tagcggtggt ttttttgttt gcaagcagca gattacgcgc
     8641 agaaaaaaag gatctcaaga agatcctttg atcttttcta cggggtctga cgctcagtgg
     8701 aacgaaaact cacgttaagg gattttggtc atgagattat caaaaaggat cttcacctag
     8761 atccttttaa attaaaaatg aagttttaaa tcaatctaaa gtatatatga gtaaacttgg
     8821 tctgacagtt accaatgctt aatcagtgag gcacctatct cagcgatctg tctatttcgt
     8881 tcatccatag ttgcctgact ccccgtcgtg tagataacta cgatacggga gggcttacca
     8941 tctggcccca gtgctgcaat gataccgcga gacccacgct caccggctcc agatttatca
     9001 gcaataaacc agccagccgg aagggccgag cgcagaagtg gtcctgcaac tttatccgcc
     9061 tccatccagt ctattaattg ttgccgggaa gctagagtaa gtagttcgcc agttaatagt
     9121 ttgcgcaacg ttgttgccat tgctacaggc atcgtggtgt cacgctcgtc gtttggtatg
     9181 gcttcattca gctccggttc ccaacgatca aggcgagtta catgatcccc catgttgtgc
     9241 aaaaaagcgg ttagctcctt cggtcctccg atcgttgtca gaagtaagtt ggccgcagtg
     9301 ttatcactca tggttatggc agcactgcat aattctctta ctgtcatgcc atccgtaaga
     9361 tgcttttctg tgactggtga gtactcaacc aagtcattct gagaatagtg tatgcggcga
     9421 ccgagttgct cttgcccggc gtcaatacgg gataataccg cgccacatag cagaacttta
     9481 aaagtgctca tcattggaaa acgttcttcg gggcgaaaac tctcaaggat cttaccgctg
     9541 ttgagatcca gttcgatgta acccactcgt gcacccaact gatcttcagc atcttttact
     9601 ttcaccagcg tttctgggtg agcaaaaaca ggaaggcaaa atgccgcaaa aaagggaata
     9661 agggcgacac ggaaatgttg aatactcata ctcttccttt ttcaatatta ttgaagcatt
     9721 tatcagggtt attgtctcat gagcggatac atatttgaat gtatttagaa aaataaacaa
     9781 ataggggttc cgcgcacatt tccccgaaaa gtgccacctg acg
//
