LOCUS       sequence-328118-        9780 bp    DNA     circular SYN 15-DEC-2025
DEFINITION  Expression of gRNA targeting multiple HLA-A, HLA-B, and HLA-C genes.
ACCESSION   .
VERSION     .
KEYWORDS    .
SOURCE      synthetic DNA construct
  ORGANISM  synthetic DNA construct
REFERENCE   1  (bases 1 to 9780)
  AUTHORS   Lee MN, Meyerson M
  TITLE     Antigen identification for HLA class I- and HLA class II-restricted 
            T cell receptors using cytokine-capturing antigen-presenting cells.
  JOURNAL   Sci Immunol. 2021 Jan 22;6(55). pii: 6/55/eabf4001. doi: 
            10.1126/sciimmunol.abf4001.
  PUBMED    33483338
REFERENCE   2  (bases 1 to 9780)
  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: "Sci 
            Immunol."; date: "2021-01-22"; volume: "6(55). pii: 6/55/eabf4001. 
            doi"; pages: " 10.1126/sciimmunol.abf4001"
COMMENT     Sequence Label: CRISPR_HLA_class_I_2
FEATURES             Location/Qualifiers
     source          1..9780
                     /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"
     promoter        730..1007
                     /label=chicken beta-actin promoter
     intron          1008..1235
                     /label=hybrid intron
                     /note="hybrid between chicken beta-actin (CBA) and minute 
                     virus of mice (MMV) introns (Gray et al., 2011)"
     regulatory      1247..1256
                     /label=Kozak sequence
                     /note="vertebrate consensus sequence for strong initiation 
                     of translation (Kozak, 1987)"
                     /regulatory_class="other"
     CDS             1256..1321
                     /codon_start=1
                     /product="three tandem FLAG(R) epitope tags, followed by an
                     enterokinase cleavage site"
                     /label=3xFLAG
                     /translation="DYKDHDGDYKDHDIDYKDDDDK"
     CDS             1328..1348
                     /codon_start=1
                     /product="nuclear localization signal of SV40 (simian virus
                     40) large T antigen"
                     /label=SV40 NLS
                     /translation="PKKKRKV"
     CDS             1373..5473
                     /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             1373..5473
                     /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             5474..5521
                     /codon_start=1
                     /product="bipartite nuclear localization signal from 
                     nucleoplasmin"
                     /label=nucleoplasmin NLS
                     /translation="KRPAATKKAGQAKKKK"
     primer_bind     complement(5549..5566)
                     /label=BGH-rev
                     /note="Bovine growth hormone terminator, reverse primer. 
                     Also called BGH reverse"
     polyA_signal    5555..5762
                     /label=bGH poly(A) signal
                     /note="bovine growth hormone polyadenylation signal"
     primer_bind     complement(5775..5795)
                     /label=pBABE 3'
                     /note="SV40 enhancer, reverse primer for pBABE vectors"
     promoter        5780..6109
                     /label=SV40 promoter
                     /note="SV40 enhancer and early promoter"
     rep_origin      5960..6095
                     /label=SV40 ori
                     /note="SV40 origin of replication"
     primer_bind     6022..6041
                     /label=SV40pro-F
                     /note="SV40 promoter/origin, forward primer"
     CDS             6127..6726
                     /codon_start=1
                     /gene="pac from Streptomyces alboniger"
                     /product="puromycin N-acetyltransferase"
                     /label=PuroR
                     /note="confers resistance to puromycin"
                     /translation="MTEYKPTVRLATRDDVPRAVRTLAAAFADYPATRHTVDPDRHIER
                     VTELQELFLTRVGLDIGKVWVADDGAAVAVWTTPESVEAGAVFAEIGPRMAELSGSRLA
                     AQQQMEGLLAPHRPKEPAWFLATVGVSPDHQGKGLGSAVVLPGVEAAERAGVPAFLETS
                     APRNLPFYERLGFTVTADVEVPEGPRTWCMTRKPGA"
     primer_bind     complement(6127..6146)
                     /label=Puro-R
                     /note="Puromycin resistance gene, reverse primer. Also 
                     called puro-variant-R"
     primer_bind     6623..6643
                     /label=Puro-F
                     /note="Puromycin resistance gene, forward primer"
     polyA_signal    6905..7026
                     /label=SV40 poly(A) signal
                     /note="SV40 polyadenylation signal"
     primer_bind     complement(6942..6961)
                     /label=SV40pA-R
                     /note="SV40 polyA, reverse primer"
     primer_bind     6996..7015
                     /label=EBV-rev
                     /note="SV40 polyA terminator, reverse primer"
     repeat_region   7043..7183
                     /label=AAV2 ITR
                     /note="inverted terminal repeat of adeno-associated virus 
                     serotype 2"
     repeat_region   7043..7172
                     /label=AAV2 ITR
     rep_origin      7258..7713
                     /direction=RIGHT
                     /label=f1 ori
                     /note="f1 bacteriophage origin of replication; arrow 
                     indicates direction of (+) strand synthesis"
     primer_bind     complement(7345..7364)
                     /label=F1ori-R
                     /note="F1 origin, reverse primer"
     primer_bind     7555..7576
                     /label=F1ori-F
                     /note="F1 origin, forward primer"
     primer_bind     complement(7730..7749)
                     /label=pRS-marker
                     /note="pRS vectors, use to sequence yeast selectable 
                     marker"
     primer_bind     7849..7871
                     /label=pGEX 3'
                     /note="pGEX vectors, reverse primer"
     primer_bind     complement(7909..7927)
                     /label=pBRforEco
                     /note="pBR322 vectors, upsteam of EcoRI site, forward 
                     primer"
     promoter        7995..8099
                     /gene="bla"
                     /label=AmpR promoter
     CDS             join(8100..8168,8169..8960)
                     /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(8318..8337)
                     /label=Amp-R
                     /note="Ampicillin resistance gene, reverse primer"
     rep_origin      9131..9719
                     /direction=RIGHT
                     /label=ori
                     /note="high-copy-number ColE1/pMB1/pBR322/pUC origin of 
                     replication"
     primer_bind     9620..9639
                     /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 cggctactac aaccagagcg 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 gcgcgcgcca ggcggggcgg ggcggggcga ggggcggggc ggggcgaggc
      901 ggagaggtgc ggcggcagcc aatcagagcg gcgcgctccg aaagtttcct tttatggcga
      961 ggcggcggcg gcggcggccc tataaaaagc gaagcgcgcg gcgggcggga gtcgctgcgc
     1021 gctgccttcg ccccgtgccc cgctccgccg ccgcctcgcg ccgcccgccc cggctctgac
     1081 tgaccgcgtt actcccacag gtgagcgggc gggacggccc ttctcctccg ggctgtaatt
     1141 agctgagcaa gaggtaaggg tttaagggat ggttggttgg tggggtatta atgtttaatt
     1201 acctggagca cctgcctgaa atcacttttt ttcaggttgg accggtgcca ccatggacta
     1261 taaggaccac gacggagact acaaggatca tgatattgat tacaaagacg atgacgataa
     1321 gatggcccca aagaagaagc ggaaggtcgg tatccacgga gtcccagcag ccgacaagaa
     1381 gtacagcatc ggcctggaca tcggcaccaa ctctgtgggc tgggccgtga tcaccgacga
     1441 gtacaaggtg cccagcaaga aattcaaggt gctgggcaac accgaccggc acagcatcaa
     1501 gaagaacctg atcggagccc tgctgttcga cagcggcgaa acagccgagg ccacccggct
     1561 gaagagaacc gccagaagaa gatacaccag acggaagaac cggatctgct atctgcaaga
     1621 gatcttcagc aacgagatgg ccaaggtgga cgacagcttc ttccacagac tggaagagtc
     1681 cttcctggtg gaagaggata agaagcacga gcggcacccc atcttcggca acatcgtgga
     1741 cgaggtggcc taccacgaga agtaccccac catctaccac ctgagaaaga aactggtgga
     1801 cagcaccgac aaggccgacc tgcggctgat ctatctggcc ctggcccaca tgatcaagtt
     1861 ccggggccac ttcctgatcg agggcgacct gaaccccgac aacagcgacg tggacaagct
     1921 gttcatccag ctggtgcaga cctacaacca gctgttcgag gaaaacccca tcaacgccag
     1981 cggcgtggac gccaaggcca tcctgtctgc cagactgagc aagagcagac ggctggaaaa
     2041 tctgatcgcc cagctgcccg gcgagaagaa gaatggcctg ttcggaaacc tgattgccct
     2101 gagcctgggc ctgaccccca acttcaagag caacttcgac ctggccgagg atgccaaact
     2161 gcagctgagc aaggacacct acgacgacga cctggacaac ctgctggccc agatcggcga
     2221 ccagtacgcc gacctgtttc tggccgccaa gaacctgtcc gacgccatcc tgctgagcga
     2281 catcctgaga gtgaacaccg agatcaccaa ggcccccctg agcgcctcta tgatcaagag
     2341 atacgacgag caccaccagg acctgaccct gctgaaagct ctcgtgcggc agcagctgcc
     2401 tgagaagtac aaagagattt tcttcgacca gagcaagaac ggctacgccg gctacattga
     2461 cggcggagcc agccaggaag agttctacaa gttcatcaag cccatcctgg aaaagatgga
     2521 cggcaccgag gaactgctcg tgaagctgaa cagagaggac ctgctgcgga agcagcggac
     2581 cttcgacaac ggcagcatcc cccaccagat ccacctggga gagctgcacg ccattctgcg
     2641 gcggcaggaa gatttttacc cattcctgaa ggacaaccgg gaaaagatcg agaagatcct
     2701 gaccttccgc atcccctact acgtgggccc tctggccagg ggaaacagca gattcgcctg
     2761 gatgaccaga aagagcgagg aaaccatcac cccctggaac ttcgaggaag tggtggacaa
     2821 gggcgcttcc gcccagagct tcatcgagcg gatgaccaac ttcgataaga acctgcccaa
     2881 cgagaaggtg ctgcccaagc acagcctgct gtacgagtac ttcaccgtgt ataacgagct
     2941 gaccaaagtg aaatacgtga ccgagggaat gagaaagccc gccttcctga gcggcgagca
     3001 gaaaaaggcc atcgtggacc tgctgttcaa gaccaaccgg aaagtgaccg tgaagcagct
     3061 gaaagaggac tacttcaaga aaatcgagtg cttcgactcc gtggaaatct ccggcgtgga
     3121 agatcggttc aacgcctccc tgggcacata ccacgatctg ctgaaaatta tcaaggacaa
     3181 ggacttcctg gacaatgagg aaaacgagga cattctggaa gatatcgtgc tgaccctgac
     3241 actgtttgag gacagagaga tgatcgagga acggctgaaa acctatgccc acctgttcga
     3301 cgacaaagtg atgaagcagc tgaagcggcg gagatacacc ggctggggca ggctgagccg
     3361 gaagctgatc aacggcatcc gggacaagca gtccggcaag acaatcctgg atttcctgaa
     3421 gtccgacggc ttcgccaaca gaaacttcat gcagctgatc cacgacgaca gcctgacctt
     3481 taaagaggac atccagaaag cccaggtgtc cggccagggc gatagcctgc acgagcacat
     3541 tgccaatctg gccggcagcc ccgccattaa gaagggcatc ctgcagacag tgaaggtggt
     3601 ggacgagctc gtgaaagtga tgggccggca caagcccgag aacatcgtga tcgaaatggc
     3661 cagagagaac cagaccaccc agaagggaca gaagaacagc cgcgagagaa tgaagcggat
     3721 cgaagagggc atcaaagagc tgggcagcca gatcctgaaa gaacaccccg tggaaaacac
     3781 ccagctgcag aacgagaagc tgtacctgta ctacctgcag aatgggcggg atatgtacgt
     3841 ggaccaggaa ctggacatca accggctgtc cgactacgat gtggaccata tcgtgcctca
     3901 gagctttctg aaggacgact ccatcgacaa caaggtgctg accagaagcg acaagaaccg
     3961 gggcaagagc gacaacgtgc cctccgaaga ggtcgtgaag aagatgaaga actactggcg
     4021 gcagctgctg aacgccaagc tgattaccca gagaaagttc gacaatctga ccaaggccga
     4081 gagaggcggc ctgagcgaac tggataaggc cggcttcatc aagagacagc tggtggaaac
     4141 ccggcagatc acaaagcacg tggcacagat cctggactcc cggatgaaca ctaagtacga
     4201 cgagaatgac aagctgatcc gggaagtgaa agtgatcacc ctgaagtcca agctggtgtc
     4261 cgatttccgg aaggatttcc agttttacaa agtgcgcgag atcaacaact accaccacgc
     4321 ccacgacgcc tacctgaacg ccgtcgtggg aaccgccctg atcaaaaagt accctaagct
     4381 ggaaagcgag ttcgtgtacg gcgactacaa ggtgtacgac gtgcggaaga tgatcgccaa
     4441 gagcgagcag gaaatcggca aggctaccgc caagtacttc ttctacagca acatcatgaa
     4501 ctttttcaag accgagatta ccctggccaa cggcgagatc cggaagcggc ctctgatcga
     4561 gacaaacggc gaaaccgggg agatcgtgtg ggataagggc cgggattttg ccaccgtgcg
     4621 gaaagtgctg agcatgcccc aagtgaatat cgtgaaaaag accgaggtgc agacaggcgg
     4681 cttcagcaaa gagtctatcc tgcccaagag gaacagcgat aagctgatcg ccagaaagaa
     4741 ggactgggac cctaagaagt acggcggctt cgacagcccc accgtggcct attctgtgct
     4801 ggtggtggcc aaagtggaaa agggcaagtc caagaaactg aagagtgtga aagagctgct
     4861 ggggatcacc atcatggaaa gaagcagctt cgagaagaat cccatcgact ttctggaagc
     4921 caagggctac aaagaagtga aaaaggacct gatcatcaag ctgcctaagt actccctgtt
     4981 cgagctggaa aacggccgga agagaatgct ggcctctgcc ggcgaactgc agaagggaaa
     5041 cgaactggcc ctgccctcca aatatgtgaa cttcctgtac ctggccagcc actatgagaa
     5101 gctgaagggc tcccccgagg ataatgagca gaaacagctg tttgtggaac agcacaagca
     5161 ctacctggac gagatcatcg agcagatcag cgagttctcc aagagagtga tcctggccga
     5221 cgctaatctg gacaaagtgc tgtccgccta caacaagcac cgggataagc ccatcagaga
     5281 gcaggccgag aatatcatcc acctgtttac cctgaccaat ctgggagccc ctgccgcctt
     5341 caagtacttt gacaccacca tcgaccggaa gaggtacacc agcaccaaag aggtgctgga
     5401 cgccaccctg atccaccaga gcatcaccgg cctgtacgag acacggatcg acctgtctca
     5461 gctgggaggc gacaaaaggc cggcggccac gaaaaaggcc ggccaggcaa aaaagaaaaa
     5521 gtaagaattc ctagagctcg ctgatcagcc tcgactgtgc cttctagttg ccagccatct
     5581 gttgtttgcc cctcccccgt gccttccttg accctggaag gtgccactcc cactgtcctt
     5641 tcctaataaa atgaggaaat tgcatcgcat tgtctgagta ggtgtcattc tattctgggg
     5701 ggtggggtgg ggcaggacag caagggggag gattgggaag agaatagcag gcatgctggg
     5761 gagcggccgc ctgtggaatg tgtgtcagtt agggtgtgga aagtccccag gctccccagc
     5821 aggcagaagt atgcaaagca tgcatctcaa ttagtcagca accaggtgtg gaaagtcccc
     5881 aggctcccca gcaggcagaa gtatgcaaag catgcatctc aattagtcag caaccatagt
     5941 cccgccccta actccgccca tcccgcccct aactccgccc agttccgccc attctccgcc
     6001 ccatggctga ctaatttttt ttatttatgc agaggccgag gccgcctctg cctctgagct
     6061 attccagaag tagtgaggag gcttttttgg aggcctaggc ttttgcaaaa agctctcgag
     6121 gccaccatga ccgagtacaa gcccacggtg cgcctcgcca cccgcgacga cgtccccagg
     6181 gccgtacgca ccctcgccgc cgcgttcgcc gactaccccg ccacgcgcca caccgtcgat
     6241 ccggaccgcc acatcgagcg ggtcaccgag ctgcaagaac tcttcctcac gcgcgtcggg
     6301 ctcgacatcg gcaaggtgtg ggtcgcggac gacggcgccg cggtggcggt ctggaccacg
     6361 ccggagagcg tcgaagcggg ggcggtgttc gccgagatcg gcccgcgcat ggccgagttg
     6421 agcggttccc ggctggccgc gcagcaacag atggaaggcc tcctggcgcc gcaccggccc
     6481 aaggagcccg cgtggttcct ggccaccgtc ggcgtctcgc ccgaccacca gggcaagggt
     6541 ctgggcagcg ccgtcgtgct ccccggagtg gaggcggccg agcgcgccgg ggtgcccgcc
     6601 ttcctggaga catccgcgcc ccgcaacctc cccttctacg agcggctcgg cttcaccgtc
     6661 accgccgacg tcgaggtgcc cgaaggaccg cgcacctggt gcatgacccg caagcccggt
     6721 gcctgactcg agcgggactc tggggttcga aatgaccgac caagcgacgc ccaacctgcc
     6781 atcacgagat ttcgattcca ccgccgcctt ctatgaaagg ttgggcttcg gaatcgtttt
     6841 ccgggacgcc ggctggatga tcctccagcg cggggatctc atgctggagt tcttcgccca
     6901 ccccaacttg tttattgcag cttataatgg ttacaaataa agcaatagca tcacaaattt
     6961 cacaaataaa gcattttttt cactgcattc tagttgtggt ttgtccaaac tcatcaatgt
     7021 atcttatcat gtctgcggcc gcaggaaccc ctagtgatgg agttggccac tccctctctg
     7081 cgcgctcgct cgctcactga ggccgggcga ccaaaggtcg cccgacgccc gggctttgcc
     7141 cgggcggcct cagtgagcga gcgagcgcgc agctgcctgc aggggcgcct gatgcggtat
     7201 tttctcctta cgcatctgtg cggtatttca caccgcatac gtcaaagcaa ccatagtacg
     7261 cgccctgtag cggcgcatta agcgcggcgg gtgtggtggt tacgcgcagc gtgaccgcta
     7321 cacttgccag cgccttagcg cccgctcctt tcgctttctt cccttccttt ctcgccacgt
     7381 tcgccggctt tccccgtcaa gctctaaatc gggggctccc tttagggttc cgatttagtg
     7441 ctttacggca cctcgacccc aaaaaacttg atttgggtga tggttcacgt agtgggccat
     7501 cgccctgata gacggttttt cgccctttga cgttggagtc cacgttcttt aatagtggac
     7561 tcttgttcca aactggaaca acactcaact ctatctcggg ctattctttt gatttataag
     7621 ggattttgcc gatttcggtc tattggttaa aaaatgagct gatttaacaa aaatttaacg
     7681 cgaattttaa caaaatatta acgtttacaa ttttatggtg cactctcagt acaatctgct
     7741 ctgatgccgc atagttaagc cagccccgac acccgccaac acccgctgac gcgccctgac
     7801 gggcttgtct gctcccggca tccgcttaca gacaagctgt gaccgtctcc gggagctgca
     7861 tgtgtcagag gttttcaccg tcatcaccga aacgcgcgag acgaaagggc ctcgtgatac
     7921 gcctattttt ataggttaat gtcatgataa taatggtttc ttagacgtca ggtggcactt
     7981 ttcggggaaa tgtgcgcgga acccctattt gtttattttt ctaaatacat tcaaatatgt
     8041 atccgctcat gagacaataa ccctgataaa tgcttcaata atattgaaaa aggaagagta
     8101 tgagtattca acatttccgt gtcgccctta ttcccttttt tgcggcattt tgccttcctg
     8161 tttttgctca cccagaaacg ctggtgaaag taaaagatgc tgaagatcag ttgggtgcac
     8221 gagtgggtta catcgaactg gatctcaaca gcggtaagat ccttgagagt tttcgccccg
     8281 aagaacgttt tccaatgatg agcactttta aagttctgct atgtggcgcg gtattatccc
     8341 gtattgacgc cgggcaagag caactcggtc gccgcataca ctattctcag aatgacttgg
     8401 ttgagtactc accagtcaca gaaaagcatc ttacggatgg catgacagta agagaattat
     8461 gcagtgctgc cataaccatg agtgataaca ctgcggccaa cttacttctg acaacgatcg
     8521 gaggaccgaa ggagctaacc gcttttttgc acaacatggg ggatcatgta actcgccttg
     8581 atcgttggga accggagctg aatgaagcca taccaaacga cgagcgtgac accacgatgc
     8641 ctgtagcaat ggcaacaacg ttgcgcaaac tattaactgg cgaactactt actctagctt
     8701 cccggcaaca attaatagac tggatggagg cggataaagt tgcaggacca cttctgcgct
     8761 cggcccttcc ggctggctgg tttattgctg ataaatctgg agccggtgag cgtggaagcc
     8821 gcggtatcat tgcagcactg gggccagatg gtaagccctc ccgtatcgta gttatctaca
     8881 cgacggggag tcaggcaact atggatgaac gaaatagaca gatcgctgag ataggtgcct
     8941 cactgattaa gcattggtaa ctgtcagacc aagtttactc atatatactt tagattgatt
     9001 taaaacttca tttttaattt aaaaggatct aggtgaagat cctttttgat aatctcatga
     9061 ccaaaatccc ttaacgtgag ttttcgttcc actgagcgtc agaccccgta gaaaagatca
     9121 aaggatcttc ttgagatcct ttttttctgc gcgtaatctg ctgcttgcaa acaaaaaaac
     9181 caccgctacc agcggtggtt tgtttgccgg atcaagagct accaactctt tttccgaagg
     9241 taactggctt cagcagagcg cagataccaa atactgttct tctagtgtag ccgtagttag
     9301 gccaccactt caagaactct gtagcaccgc ctacatacct cgctctgcta atcctgttac
     9361 cagtggctgc tgccagtggc gataagtcgt gtcttaccgg gttggactca agacgatagt
     9421 taccggataa ggcgcagcgg tcgggctgaa cggggggttc gtgcacacag cccagcttgg
     9481 agcgaacgac ctacaccgaa ctgagatacc tacagcgtga gctatgagaa agcgccacgc
     9541 ttcccgaagg gagaaaggcg gacaggtatc cggtaagcgg cagggtcgga acaggagagc
     9601 gcacgaggga gcttccaggg ggaaacgcct ggtatcttta tagtcctgtc gggtttcgcc
     9661 acctctgact tgagcgtcga tttttgtgat gctcgtcagg ggggcggagc ctatggaaaa
     9721 acgccagcaa cgcggccttt ttacggttcc tggccttttg ctggcctttt gctcacatgt
//
