LOCUS       sequence-361331-        9137 bp    DNA     circular SYN 15-DEC-2025
DEFINITION  Cas9 from S.pyogenes with 2A-Puro, and the 2B-sgRNA to targeting 
            exon 2 of human HLA-G gene.
ACCESSION   .
VERSION     .
KEYWORDS    .
SOURCE      synthetic DNA construct
  ORGANISM  synthetic DNA construct
REFERENCE   1  (bases 1 to 9137)
  AUTHORS   Palma MB, Tronik-Le Roux D, Amin G, Castaneda S, Mobbs AM, Scarafia 
            MA, La Greca A, Daouya M, Poras I, Inda AM, Moro LN, Carosella ED, 
            Garcia MN, Miriuka SG
  TITLE     HLA-G gene editing in tumor cell lines as a novel alternative in 
            cancer immunotherapy.
  JOURNAL   Sci Rep. 2021 Nov 12;11(1):22158. doi: 10.1038/s41598-021-01572-0.
  PUBMED    34773056
REFERENCE   2  (bases 1 to 9137)
  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 Rep."; 
            date: "2021-11-12"; volume: "11(1):22158. doi"; pages: " 
            10.1038/s41598-021-01572-0"
COMMENT     Sequence Label: pSpCas9-Puro HLAG-2B-sgRNA
FEATURES             Location/Qualifiers
     source          1..9137
                     /mol_type="other DNA"
                     /organism="synthetic DNA construct"
     intron          122..349
                     /label=hybrid intron
                     /note="hybrid between chicken beta-actin (CBA) and minute 
                     virus of mice (MMV) introns (Gray et al., 2011)"
     regulatory      361..370
                     /label=Kozak sequence
                     /note="vertebrate consensus sequence for strong initiation 
                     of translation (Kozak, 1987)"
                     /regulatory_class="other"
     CDS             370..435
                     /codon_start=1
                     /product="three tandem FLAG(R) epitope tags, followed by an
                     enterokinase cleavage site"
                     /label=3xFLAG
                     /translation="DYKDHDGDYKDHDIDYKDDDDK"
     CDS             442..462
                     /codon_start=1
                     /product="nuclear localization signal of SV40 (simian virus
                     40) large T antigen"
                     /label=SV40 NLS
                     /translation="PKKKRKV"
     CDS             487..4587
                     /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             487..4587
                     /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             4588..4635
                     /codon_start=1
                     /product="bipartite nuclear localization signal from 
                     nucleoplasmin"
                     /label=nucleoplasmin NLS
                     /translation="KRPAATKKAGQAKKKK"
     CDS             4651..4704
                     /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             4705..5304
                     /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(4705..4724)
                     /label=Puro-R
                     /note="Puromycin resistance gene, reverse primer. Also 
                     called puro-variant-R"
     primer_bind     5201..5221
                     /label=Puro-F
                     /note="Puromycin resistance gene, forward primer"
     primer_bind     complement(5332..5349)
                     /label=BGH-rev
                     /note="Bovine growth hormone terminator, reverse primer. 
                     Also called BGH reverse"
     polyA_signal    5338..5545
                     /label=bGH poly(A) signal
                     /note="bovine growth hormone polyadenylation signal"
     repeat_region   5554..5694
                     /label=AAV2 ITR
                     /note="inverted terminal repeat of adeno-associated virus 
                     serotype 2"
     repeat_region   5554..5683
                     /label=AAV2 ITR
     rep_origin      5769..6224
                     /direction=RIGHT
                     /label=f1 ori
                     /note="f1 bacteriophage origin of replication; arrow 
                     indicates direction of (+) strand synthesis"
     primer_bind     complement(5856..5875)
                     /label=F1ori-R
                     /note="F1 origin, reverse primer"
     primer_bind     6066..6087
                     /label=F1ori-F
                     /note="F1 origin, forward primer"
     primer_bind     complement(6241..6260)
                     /label=pRS-marker
                     /note="pRS vectors, use to sequence yeast selectable 
                     marker"
     primer_bind     6360..6382
                     /label=pGEX 3'
                     /note="pGEX vectors, reverse primer"
     primer_bind     complement(6420..6438)
                     /label=pBRforEco
                     /note="pBR322 vectors, upsteam of EcoRI site, forward 
                     primer"
     promoter        6506..6610
                     /gene="bla"
                     /label=AmpR promoter
     CDS             join(6611..6679,6680..7471)
                     /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(6829..6848)
                     /label=Amp-R
                     /note="Ampicillin resistance gene, reverse primer"
     rep_origin      7642..8230
                     /direction=RIGHT
                     /label=ori
                     /note="high-copy-number ColE1/pMB1/pBR322/pUC origin of 
                     replication"
     primer_bind     8131..8150
                     /label=pBR322ori-F
                     /note="pBR322 origin, forward primer"
     promoter        8292..8540
                     /label=U6 promoter
                     /note="RNA polymerase III promoter for human U6 snRNA 
                     (Domitrovich & Kunkel, 2003)"
     primer_bind     8292..8312
                     /label=hU6-F
                     /note="Human U6 promoter, forward primer"
     primer_bind     8463..8482
                     /label=LKO.1 5'
                     /note="Human U6 promoter, forward primer"
     misc_RNA        8561..8636
                     /label=gRNA scaffold
                     /note="guide RNA scaffold for the Streptococcus pyogenes 
                     CRISPR/Cas9 system"
     enhancer        8733..9018
                     /label=CMV enhancer
                     /note="human cytomegalovirus immediate early enhancer; 
                     contains an 18-bp deletion relative to the standard CMV 
                     enhancer"
ORIGIN
        1 gggcggggcg aggcggagag gtgcggcggc agccaatcag agcggcgcgc tccgaaagtt
       61 tccttttatg gcgaggcggc ggcggcggcg gccctataaa aagcgaagcg cgcggcgggc
      121 gggagtcgct gcgcgctgcc ttcgccccgt gccccgctcc gccgccgcct cgcgccgccc
      181 gccccggctc tgactgaccg cgttactccc acaggtgagc gggcgggacg gcccttctcc
      241 tccgggctgt aattagctga gcaagaggta agggtttaag ggatggttgg ttggtggggt
      301 attaatgttt aattacctgg agcacctgcc tgaaatcact ttttttcagg ttggaccggt
      361 gccaccatgg actataagga ccacgacgga gactacaagg atcatgatat tgattacaaa
      421 gacgatgacg ataagatggc cccaaagaag aagcggaagg tcggtatcca cggagtccca
      481 gcagccgaca agaagtacag catcggcctg gacatcggca ccaactctgt gggctgggcc
      541 gtgatcaccg acgagtacaa ggtgcccagc aagaaattca aggtgctggg caacaccgac
      601 cggcacagca tcaagaagaa cctgatcgga gccctgctgt tcgacagcgg cgaaacagcc
      661 gaggccaccc ggctgaagag aaccgccaga agaagataca ccagacggaa gaaccggatc
      721 tgctatctgc aagagatctt cagcaacgag atggccaagg tggacgacag cttcttccac
      781 agactggaag agtccttcct ggtggaagag gataagaagc acgagcggca ccccatcttc
      841 ggcaacatcg tggacgaggt ggcctaccac gagaagtacc ccaccatcta ccacctgaga
      901 aagaaactgg tggacagcac cgacaaggcc gacctgcggc tgatctatct ggccctggcc
      961 cacatgatca agttccgggg ccacttcctg atcgagggcg acctgaaccc cgacaacagc
     1021 gacgtggaca agctgttcat ccagctggtg cagacctaca accagctgtt cgaggaaaac
     1081 cccatcaacg ccagcggcgt ggacgccaag gccatcctgt ctgccagact gagcaagagc
     1141 agacggctgg aaaatctgat cgcccagctg cccggcgaga agaagaatgg cctgttcgga
     1201 aacctgattg ccctgagcct gggcctgacc cccaacttca agagcaactt cgacctggcc
     1261 gaggatgcca aactgcagct gagcaaggac acctacgacg acgacctgga caacctgctg
     1321 gcccagatcg gcgaccagta cgccgacctg tttctggccg ccaagaacct gtccgacgcc
     1381 atcctgctga gcgacatcct gagagtgaac accgagatca ccaaggcccc cctgagcgcc
     1441 tctatgatca agagatacga cgagcaccac caggacctga ccctgctgaa agctctcgtg
     1501 cggcagcagc tgcctgagaa gtacaaagag attttcttcg accagagcaa gaacggctac
     1561 gccggctaca ttgacggcgg agccagccag gaagagttct acaagttcat caagcccatc
     1621 ctggaaaaga tggacggcac cgaggaactg ctcgtgaagc tgaacagaga ggacctgctg
     1681 cggaagcagc ggaccttcga caacggcagc atcccccacc agatccacct gggagagctg
     1741 cacgccattc tgcggcggca ggaagatttt tacccattcc tgaaggacaa ccgggaaaag
     1801 atcgagaaga tcctgacctt ccgcatcccc tactacgtgg gccctctggc caggggaaac
     1861 agcagattcg cctggatgac cagaaagagc gaggaaacca tcaccccctg gaacttcgag
     1921 gaagtggtgg acaagggcgc ttccgcccag agcttcatcg agcggatgac caacttcgat
     1981 aagaacctgc ccaacgagaa ggtgctgccc aagcacagcc tgctgtacga gtacttcacc
     2041 gtgtataacg agctgaccaa agtgaaatac gtgaccgagg gaatgagaaa gcccgccttc
     2101 ctgagcggcg agcagaaaaa ggccatcgtg gacctgctgt tcaagaccaa ccggaaagtg
     2161 accgtgaagc agctgaaaga ggactacttc aagaaaatcg agtgcttcga ctccgtggaa
     2221 atctccggcg tggaagatcg gttcaacgcc tccctgggca cataccacga tctgctgaaa
     2281 attatcaagg acaaggactt cctggacaat gaggaaaacg aggacattct ggaagatatc
     2341 gtgctgaccc tgacactgtt tgaggacaga gagatgatcg aggaacggct gaaaacctat
     2401 gcccacctgt tcgacgacaa agtgatgaag cagctgaagc ggcggagata caccggctgg
     2461 ggcaggctga gccggaagct gatcaacggc atccgggaca agcagtccgg caagacaatc
     2521 ctggatttcc tgaagtccga cggcttcgcc aacagaaact tcatgcagct gatccacgac
     2581 gacagcctga cctttaaaga ggacatccag aaagcccagg tgtccggcca gggcgatagc
     2641 ctgcacgagc acattgccaa tctggccggc agccccgcca ttaagaaggg catcctgcag
     2701 acagtgaagg tggtggacga gctcgtgaaa gtgatgggcc ggcacaagcc cgagaacatc
     2761 gtgatcgaaa tggccagaga gaaccagacc acccagaagg gacagaagaa cagccgcgag
     2821 agaatgaagc ggatcgaaga gggcatcaaa gagctgggca gccagatcct gaaagaacac
     2881 cccgtggaaa acacccagct gcagaacgag aagctgtacc tgtactacct gcagaatggg
     2941 cgggatatgt acgtggacca ggaactggac atcaaccggc tgtccgacta cgatgtggac
     3001 catatcgtgc ctcagagctt tctgaaggac gactccatcg acaacaaggt gctgaccaga
     3061 agcgacaaga accggggcaa gagcgacaac gtgccctccg aagaggtcgt gaagaagatg
     3121 aagaactact ggcggcagct gctgaacgcc aagctgatta cccagagaaa gttcgacaat
     3181 ctgaccaagg ccgagagagg cggcctgagc gaactggata aggccggctt catcaagaga
     3241 cagctggtgg aaacccggca gatcacaaag cacgtggcac agatcctgga ctcccggatg
     3301 aacactaagt acgacgagaa tgacaagctg atccgggaag tgaaagtgat caccctgaag
     3361 tccaagctgg tgtccgattt ccggaaggat ttccagtttt acaaagtgcg cgagatcaac
     3421 aactaccacc acgcccacga cgcctacctg aacgccgtcg tgggaaccgc cctgatcaaa
     3481 aagtacccta agctggaaag cgagttcgtg tacggcgact acaaggtgta cgacgtgcgg
     3541 aagatgatcg ccaagagcga gcaggaaatc ggcaaggcta ccgccaagta cttcttctac
     3601 agcaacatca tgaacttttt caagaccgag attaccctgg ccaacggcga gatccggaag
     3661 cggcctctga tcgagacaaa cggcgaaacc ggggagatcg tgtgggataa gggccgggat
     3721 tttgccaccg tgcggaaagt gctgagcatg ccccaagtga atatcgtgaa aaagaccgag
     3781 gtgcagacag gcggcttcag caaagagtct atcctgccca agaggaacag cgataagctg
     3841 atcgccagaa agaaggactg ggaccctaag aagtacggcg gcttcgacag ccccaccgtg
     3901 gcctattctg tgctggtggt ggccaaagtg gaaaagggca agtccaagaa actgaagagt
     3961 gtgaaagagc tgctggggat caccatcatg gaaagaagca gcttcgagaa gaatcccatc
     4021 gactttctgg aagccaaggg ctacaaagaa gtgaaaaagg acctgatcat caagctgcct
     4081 aagtactccc tgttcgagct ggaaaacggc cggaagagaa tgctggcctc tgccggcgaa
     4141 ctgcagaagg gaaacgaact ggccctgccc tccaaatatg tgaacttcct gtacctggcc
     4201 agccactatg agaagctgaa gggctccccc gaggataatg agcagaaaca gctgtttgtg
     4261 gaacagcaca agcactacct ggacgagatc atcgagcaga tcagcgagtt ctccaagaga
     4321 gtgatcctgg ccgacgctaa tctggacaaa gtgctgtccg cctacaacaa gcaccgggat
     4381 aagcccatca gagagcaggc cgagaatatc atccacctgt ttaccctgac caatctggga
     4441 gcccctgccg ccttcaagta ctttgacacc accatcgacc ggaagaggta caccagcacc
     4501 aaagaggtgc tggacgccac cctgatccac cagagcatca ccggcctgta cgagacacgg
     4561 atcgacctgt ctcagctggg aggcgacaaa aggccggcgg ccacgaaaaa ggccggccag
     4621 gcaaaaaaga aaaaggaatt cggcagtgga gagggcagag gaagtctgct aacatgcggt
     4681 gacgtcgagg agaatcctgg cccaatgacc gagtacaagc ccacggtgcg cctcgccacc
     4741 cgcgacgacg tccccagggc cgtacgcacc ctcgccgccg cgttcgccga ctaccccgcc
     4801 acgcgccaca ccgtcgatcc ggaccgccac atcgagcggg tcaccgagct gcaagaactc
     4861 ttcctcacgc gcgtcgggct cgacatcggc aaggtgtggg tcgcggacga cggcgccgcg
     4921 gtggcggtct ggaccacgcc ggagagcgtc gaagcggggg cggtgttcgc cgagatcggc
     4981 ccgcgcatgg ccgagttgag cggttcccgg ctggccgcgc agcaacagat ggaaggcctc
     5041 ctggcgccgc accggcccaa ggagcccgcg tggttcctgg ccaccgtcgg agtctcgccc
     5101 gaccaccagg gcaagggtct gggcagcgcc gtcgtgctcc ccggagtgga ggcggccgag
     5161 cgcgccgggg tgcccgcctt cctggagacc tccgcgcccc gcaacctccc cttctacgag
     5221 cggctcggct tcaccgtcac cgccgacgtc gaggtgcccg aaggaccgcg cacctggtgc
     5281 atgacccgca agcccggtgc ctgagaattc taactagagc tcgctgatca gcctcgactg
     5341 tgccttctag ttgccagcca tctgttgttt gcccctcccc cgtgccttcc ttgaccctgg
     5401 aaggtgccac tcccactgtc ctttcctaat aaaatgagga aattgcatcg cattgtctga
     5461 gtaggtgtca ttctattctg gggggtgggg tggggcagga cagcaagggg gaggattggg
     5521 aagagaatag caggcatgct ggggagcggc cgcaggaacc cctagtgatg gagttggcca
     5581 ctccctctct gcgcgctcgc tcgctcactg aggccgggcg accaaaggtc gcccgacgcc
     5641 cgggctttgc ccgggcggcc tcagtgagcg agcgagcgcg cagctgcctg caggggcgcc
     5701 tgatgcggta ttttctcctt acgcatctgt gcggtatttc acaccgcata cgtcaaagca
     5761 accatagtac gcgccctgta gcggcgcatt aagcgcggcg ggtgtggtgg ttacgcgcag
     5821 cgtgaccgct acacttgcca gcgccttagc gcccgctcct ttcgctttct tcccttcctt
     5881 tctcgccacg ttcgccggct ttccccgtca agctctaaat cgggggctcc ctttagggtt
     5941 ccgatttagt gctttacggc acctcgaccc caaaaaactt gatttgggtg atggttcacg
     6001 tagtgggcca tcgccctgat agacggtttt tcgccctttg acgttggagt ccacgttctt
     6061 taatagtgga ctcttgttcc aaactggaac aacactcaac tctatctcgg gctattcttt
     6121 tgatttataa gggattttgc cgatttcggt ctattggtta aaaaatgagc tgatttaaca
     6181 aaaatttaac gcgaatttta acaaaatatt aacgtttaca attttatggt gcactctcag
     6241 tacaatctgc tctgatgccg catagttaag ccagccccga cacccgccaa cacccgctga
     6301 cgcgccctga cgggcttgtc tgctcccggc atccgcttac agacaagctg tgaccgtctc
     6361 cgggagctgc atgtgtcaga ggttttcacc gtcatcaccg aaacgcgcga gacgaaaggg
     6421 cctcgtgata cgcctatttt tataggttaa tgtcatgata ataatggttt cttagacgtc
     6481 aggtggcact tttcggggaa atgtgcgcgg aacccctatt tgtttatttt tctaaataca
     6541 ttcaaatatg tatccgctca tgagacaata accctgataa atgcttcaat aatattgaaa
     6601 aaggaagagt atgagtattc aacatttccg tgtcgccctt attccctttt ttgcggcatt
     6661 ttgccttcct gtttttgctc acccagaaac gctggtgaaa gtaaaagatg ctgaagatca
     6721 gttgggtgca cgagtgggtt acatcgaact ggatctcaac agcggtaaga tccttgagag
     6781 ttttcgcccc gaagaacgtt ttccaatgat gagcactttt aaagttctgc tatgtggcgc
     6841 ggtattatcc cgtattgacg ccgggcaaga gcaactcggt cgccgcatac actattctca
     6901 gaatgacttg gttgagtact caccagtcac agaaaagcat cttacggatg gcatgacagt
     6961 aagagaatta tgcagtgctg ccataaccat gagtgataac actgcggcca acttacttct
     7021 gacaacgatc ggaggaccga aggagctaac cgcttttttg cacaacatgg gggatcatgt
     7081 aactcgcctt gatcgttggg aaccggagct gaatgaagcc ataccaaacg acgagcgtga
     7141 caccacgatg cctgtagcaa tggcaacaac gttgcgcaaa ctattaactg gcgaactact
     7201 tactctagct tcccggcaac aattaataga ctggatggag gcggataaag ttgcaggacc
     7261 acttctgcgc tcggcccttc cggctggctg gtttattgct gataaatctg gagccggtga
     7321 gcgtggaagc cgcggtatca ttgcagcact ggggccagat ggtaagccct cccgtatcgt
     7381 agttatctac acgacgggga gtcaggcaac tatggatgaa cgaaatagac agatcgctga
     7441 gataggtgcc tcactgatta agcattggta actgtcagac caagtttact catatatact
     7501 ttagattgat ttaaaacttc atttttaatt taaaaggatc taggtgaaga tcctttttga
     7561 taatctcatg accaaaatcc cttaacgtga gttttcgttc cactgagcgt cagaccccgt
     7621 agaaaagatc aaaggatctt cttgagatcc tttttttctg cgcgtaatct gctgcttgca
     7681 aacaaaaaaa ccaccgctac cagcggtggt ttgtttgccg gatcaagagc taccaactct
     7741 ttttccgaag gtaactggct tcagcagagc gcagatacca aatactgttc ttctagtgta
     7801 gccgtagtta ggccaccact tcaagaactc tgtagcaccg cctacatacc tcgctctgct
     7861 aatcctgtta ccagtggctg ctgccagtgg cgataagtcg tgtcttaccg ggttggactc
     7921 aagacgatag ttaccggata aggcgcagcg gtcgggctga acggggggtt cgtgcacaca
     7981 gcccagcttg gagcgaacga cctacaccga actgagatac ctacagcgtg agctatgaga
     8041 aagcgccacg cttcccgaag ggagaaaggc ggacaggtat ccggtaagcg gcagggtcgg
     8101 aacaggagag cgcacgaggg agcttccagg gggaaacgcc tggtatcttt atagtcctgt
     8161 cgggtttcgc cacctctgac ttgagcgtcg atttttgtga tgctcgtcag gggggcggag
     8221 cctatggaaa aacgccagca acgcggcctt tttacggttc ctggcctttt gctggccttt
     8281 tgctcacatg tgagggccta tttcccatga ttccttcata tttgcatata cgatacaagg
     8341 ctgttagaga gataattgga attaatttga ctgtaaacac aaagatatta gtacaaaata
     8401 cgtgacgtag aaagtaataa tttcttgggt agtttgcagt tttaaaatta tgttttaaaa
     8461 tggactatca tatgcttacc gtaacttgaa agtatttcga tttcttggct ttatatatct
     8521 tgtggaaagg acgaaacacc ggactttaga accaggaccg gttttagagc tagaaatagc
     8581 aagttaaaat aaggctagtc cgttatcaac ttgaaaaagt ggcaccgagt cggtgctttt
     8641 ttgttttaga gctagaaata gcaagttaaa ataaggctag tccgttttta gcgcgtgcgc
     8701 caattctgca gacaaatggc tctagaggta cccgttacat aacttacggt aaatggcccg
     8761 cctggctgac cgcccaacga cccccgccca ttgacgtcaa tagtaacgcc aatagggact
     8821 ttccattgac gtcaatgggt ggagtattta cggtaaactg cccacttggc agtacatcaa
     8881 gtgtatcata tgccaagtac gccccctatt gacgtcaatg acggtaaatg gcccgcctgg
     8941 cattgtgccc agtacatgac cttatgggac tttcctactt ggcagtacat ctacgtatta
     9001 gtcatcgcta ttaccatggt cgaggtgagc cccacgttct gcttcactct ccccatctcc
     9061 cccccctccc cacccccaat tttgtattta tttatttttt aattattttg tgcagcgatg
     9121 ggggcggggg ggggggg
//
