LOCUS       sequence-206308-        9825 bp    DNA     circular SYN 14-APR-2025
DEFINITION  Expresses sgRNA cassette (BsmBI cloning site) and EF1-A driven 
            eSpCas9 linked via P2A with mRuby2 and dominant negative mtp53 for 
            enhanced survival of hES after DSB.
ACCESSION   .
VERSION     .
KEYWORDS    .
SOURCE      synthetic DNA construct
  ORGANISM  synthetic DNA construct
REFERENCE   1  (bases 1 to 9825)
  AUTHORS   Ang LT, Tan AKY, Autio MI, Goh SH, Choo SH, Lee KL, Tan J, Pan B, 
            Lee JJH, Lum JJ, Lim CYY, Yeo IKX, Wong CJY, Liu M, Oh JLL, Chia 
            CPL, Loh CH, Chen A, Chen Q, Weissman IL, Loh KM, Lim B
  TITLE     A Roadmap for Human Liver Differentiation from Pluripotent Stem 
            Cells.
  JOURNAL   Cell Rep. 2018 Feb 20;22(8):2190-2205. doi: 
            10.1016/j.celrep.2018.01.087.
  PUBMED    29466743
REFERENCE   2  (bases 1 to 9825)
  AUTHORS   .
  TITLE     Direct Submission
  JOURNAL   Exported Apr 14, 2025 from SnapGene Server 8.0.1
            https://www.snapgene.com
COMMENT     SGRef: number: 1; type: "Journal Article"; doi: 
            "10.1016/j.celrep.2018.01"; journalName: "Cell Rep"; date: 
            "2018-02-20- 20"; volume: "22"; issue: "8"; pages: "2190-2205"
COMMENT     Sequence Label: pMIA3
FEATURES             Location/Qualifiers
     source          1..9825
                     /mol_type="other DNA"
                     /organism="synthetic DNA construct"
     promoter        39..287
                     /label=U6 promoter
                     /note="RNA polymerase III promoter for human U6 snRNA 
                     (Domitrovich & Kunkel, 2003)"
     primer_bind     39..59
                     /label=hU6-F
                     /note="Human U6 promoter, forward primer"
     primer_bind     210..229
                     /label=LKO.1 5'
                     /note="Human U6 promoter, forward primer"
     misc_RNA        304..379
                     /label=gRNA scaffold
                     /note="guide RNA scaffold for the Streptococcus pyogenes 
                     CRISPR/Cas9 system"
     promoter        492..703
                     /label=EF-1-alpha core promoter
                     /note="core promoter for human elongation factor 
                     EF-1-alpha"
     intron          704..1642
                     /label=EF-1-alpha intron A
                     /note="intron upstream of the start codon of human 
                     EF-1-alpha"
     primer_bind     1599..1619
                     /label=EF1a-F
                     /note="Human elongation factor-1a promoter, forward primer"
     regulatory      1683..1692
                     /label=Kozak sequence
                     /note="vertebrate consensus sequence for strong initiation 
                     of translation (Kozak, 1987)"
                     /regulatory_class="other"
     CDS             1692..1757
                     /codon_start=1
                     /product="three tandem FLAG(R) epitope tags, followed by an
                     enterokinase cleavage site"
                     /label=3xFLAG
                     /translation="DYKDHDGDYKDHDIDYKDDDDK"
     CDS             1764..1784
                     /codon_start=1
                     /product="nuclear localization signal of SV40 (simian virus
                     40) large T antigen"
                     /label=SV40 NLS
                     /translation="PKKKRKV"
     CDS             join(1809..4346,4347..4349,4350..4811,4812..4814,4815..4982,
                     4983..4985,4986..5909)
                     /codon_start=1
                     /product="Cas9 endonuclease from the Streptococcus pyogenes
                     Type II CRISPR/Cas system, mutated to improve targeting 
                     specificity (Slaymaker et al., 2016)"
                     /label=eSpCas9(1.1)
                     /note="carries the mutations K848A, K1003A, and R1060A"
                     /translation="DKKYSIGLDIGTNSVGWAVITDEYKVPSKKFKVLGNTDRHSIKKN
                     LIGALLFDSGETAEATRLKRTARRRYTRRKNRICYLQEIFSNEMAKVDDSFFHRLEESF
                     LVEEDKKHERHPIFGNIVDEVAYHEKYPTIYHLRKKLVDSTDKADLRLIYLALAHMIKF
                     RGHFLIEGDLNPDNSDVDKLFIQLVQTYNQLFEENPINASGVDAKAILSARLSKSRRLE
                     NLIAQLPGEKKNGLFGNLIALSLGLTPNFKSNFDLAEDAKLQLSKDTYDDDLDNLLAQI
                     GDQYADLFLAAKNLSDAILLSDILRVNTEITKAPLSASMIKRYDEHHQDLTLLKALVRQ
                     QLPEKYKEIFFDQSKNGYAGYIDGGASQEEFYKFIKPILEKMDGTEELLVKLNREDLLR
                     KQRTFDNGSIPHQIHLGELHAILRRQEDFYPFLKDNREKIEKILTFRIPYYVGPLARGN
                     SRFAWMTRKSEETITPWNFEEVVDKGASAQSFIERMTNFDKNLPNEKVLPKHSLLYEYF
                     TVYNELTKVKYVTEGMRKPAFLSGEQKKAIVDLLFKTNRKVTVKQLKEDYFKKIECFDS
                     VEISGVEDRFNASLGTYHDLLKIIKDKDFLDNEENEDILEDIVLTLTLFEDREMIEERL
                     KTYAHLFDDKVMKQLKRRRYTGWGRLSRKLINGIRDKQSGKTILDFLKSDGFANRNFMQ
                     LIHDDSLTFKEDIQKAQVSGQGDSLHEHIANLAGSPAIKKGILQTVKVVDELVKVMGRH
                     KPENIVIEMARENQTTQKGQKNSRERMKRIEEGIKELGSQILKEHPVENTQLQNEKLYL
                     YYLQNGRDMYVDQELDINRLSDYDVDHIVPQSFLADDSIDNKVLTRSDKNRGKSDNVPS
                     EEVVKKMKNYWRQLLNAKLITQRKFDNLTKAERGGLSELDKAGFIKRQLVETRQITKHV
                     AQILDSRMNTKYDENDKLIREVKVITLKSKLVSDFRKDFQFYKVREINNYHHAHDAYLN
                     AVVGTALIKKYPALESEFVYGDYKVYDVRKMIAKSEQEIGKATAKYFFYSNIMNFFKTE
                     ITLANGEIRKAPLIETNGETGEIVWDKGRDFATVRKVLSMPQVNIVKKTEVQTGGFSKE
                     SILPKRNSDKLIARKKDWDPKKYGGFDSPTVAYSVLVVAKVEKGKSKKLKSVKELLGIT
                     IMERSSFEKNPIDFLEAKGYKEVKKDLIIKLPKYSLFELENGRKRMLASAGELQKGNEL
                     ALPSKYVNFLYLASHYEKLKGSPEDNEQKQLFVEQHKHYLDEIIEQISEFSKRVILADA
                     NLDKVLSAYNKHRDKPIREQAENIIHLFTLTNLGAPAAFKYFDTTIDRKRYTSTKEVLD
                     ATLIHQSITGLYETRIDLSQLGGD"
     CDS             5913..5933
                     /codon_start=1
                     /product="nuclear localization signal of SV40 (simian virus
                     40) large T antigen"
                     /label=SV40 NLS
                     /translation="PKKKRKV"
     CDS             5952..6008
                     /codon_start=1
                     /product="2A peptide from porcine teschovirus-1 
                     polyprotein"
                     /label=P2A
                     /note="Eukaryotic ribosomes fail to insert a peptide bond 
                     between the Gly and Pro residues, yielding separate 
                     polypeptides."
                     /translation="ATNFSLLKQAGDVEENPGP"
     CDS             6009..6719
                     /codon_start=1
                     /product="monomeric red fluorescent protein derived from 
                     mRuby, with improved photophysical properties (Lam et al., 
                     2012)"
                     /label=mRuby2
                     /translation="MVSKGEELIKENMRMKVVMEGSVNGHQFKCTGEGEGNPYMGTQTM
                     RIKVIEGGPLPFAFDILATSFMYGSRTFIKYPKGIPDFFKQSFPEGFTWERVTRYEDGG
                     VVTVMQDTSLEDGCLVYHVQVRGVNFPSNGPVMQKKTKGWEPNTEMMYPADGGLRGYTH
                     MALKVDGGGHLSCSFVTTYRSKKTVGNIKMPGIHAVDHRLERLEESDNEMFVVQREHAV
                     AKFAGLGGGMDELYK"
     CDS             6729..6785
                     /codon_start=1
                     /product="2A peptide from porcine teschovirus-1 
                     polyprotein"
                     /label=P2A
                     /note="Eukaryotic ribosomes fail to insert a peptide bond 
                     between the Gly and Pro residues, yielding separate 
                     polypeptides."
                     /translation="ATNFSLLKQAGDVEENPGP"
     primer_bind     complement(7122..7139)
                     /label=BGH-rev
                     /note="Bovine growth hormone terminator, reverse primer. 
                     Also called BGH reverse"
     polyA_signal    7128..7335
                     /label=bGH poly(A) signal
                     /note="bovine growth hormone polyadenylation signal"
     repeat_region   7344..7484
                     /label=AAV2 ITR
                     /note="inverted terminal repeat of adeno-associated virus 
                     serotype 2"
     repeat_region   7344..7473
                     /label=AAV2 ITR
     rep_origin      7559..7987
                     /direction=RIGHT
                     /label=f1 ori
                     /note="f1 bacteriophage origin of replication; arrow 
                     indicates direction of (+) strand synthesis"
     primer_bind     complement(7646..7665)
                     /label=F1ori-R
                     /note="F1 origin, reverse primer"
     primer_bind     7856..7877
                     /label=F1ori-F
                     /note="F1 origin, forward primer"
     promoter        8031..8135
                     /gene="bla"
                     /label=AmpR promoter
     CDS             join(8136..8204,8205..8996)
                     /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(8354..8373)
                     /label=Amp-R
                     /note="Ampicillin resistance gene, reverse primer"
     rep_origin      9167..9755
                     /direction=RIGHT
                     /label=ori
                     /note="high-copy-number ColE1/pMB1/pBR322/pUC origin of 
                     replication"
     primer_bind     9656..9675
                     /label=pBR322ori-F
                     /note="pBR322 origin, forward primer"
ORIGIN
        1 ccggccagat ctgagctcgc ggccgcgata tcgctagcga gggcctattt cccatgattc
       61 cttcatattt gcatatacga tacaaggctg ttagagagat aattggaatt aatttgactg
      121 taaacacaaa gatattagta caaaatacgt gacgtagaaa gtaataattt cttgggtagt
      181 ttgcagtttt aaaattatgt tttaaaatgg actatcatat gcttaccgta acttgaaagt
      241 atttcgattt cttggcttta tatatcttgt ggaaaggacg aaacaccgga gacggacgtc
      301 tctgttttag agctagaaat agcaagttaa aataaggcta gtccgttatc aacttgaaaa
      361 agtggcaccg agtcggtgct tttttgtttt agagctagaa atagcaagtt aaaataaggc
      421 tagtccgttt ttagcgcgtg cgccaattct gcagacaaat ggctctagat tcactcgagg
      481 tgcccgtcag tgggcagagc gcacatcgcc cacagtcccc gagaagttgg ggggaggggt
      541 cggcaattga accggtgcct agagaaggtg gcgcggggta aactgggaaa gtgatgtcgt
      601 gtactggctc cgcctttttc ccgagggtgg gggagaaccg tatataagtg cagtagtcgc
      661 cgtgaacgtt ctttttcgca acgggtttgc cgccagaaca caggtaagtg ccgtgtgtgg
      721 ttcccgcggg cctggcctct ttacgggtta tggcccttgc gtgccttgaa ttacttccac
      781 ctggctgcag tacgtgattc ttgatcccga gcttcgggtt ggaagtgggt gggagagttc
      841 gaggccttgc gcttaaggag ccccttcgcc tcgtgcttga gttgaggcct ggcctgggcg
      901 ctggggccgc cgcgtgcgaa tctggtggca ccttcgcgcc tgtctcgctg ctttcgataa
      961 gtctctagcc atttaaaatt tttgatgacc tgctgcgacg ctttttttct ggcaagatag
     1021 tcttgtaaat gcgggccaag atctgcacac tggtatttcg gtttttgggg ccgcgggcgg
     1081 cgacggggcc cgtgcgtccc agcgcacatg ttcggcgagg cggggcctgc gagcgcggcc
     1141 accgagaatc ggacgggggt agtctcaagc tggccggcct gctctggtgc ctggcctcgc
     1201 gccgccgtgt atcgccccgc cctgggcggc aaggctggcc cggtcggcac cagttgcgtg
     1261 agcggaaaga tggccgcttc ccggccctgc tgcagggagc tcaaaatgga ggacgcggcg
     1321 ctcgggagag cgggcgggtg agtcacccac acaaaggaaa agggcctttc cgtcctcagc
     1381 cgtcgcttca tgtgactcca cggagtaccg ggcgccgtcc aggcacctcg attagttctc
     1441 gagcttttgg agtacgtcgt ctttaggttg gggggagggg ttttatgcga tggagtttcc
     1501 ccacactgag tgggtggaga ctgaagttag gccagcttgg cacttgatgt aattctcctt
     1561 ggaatttgcc ctttttgagt ttggatcttg gttcattctc aagcctcaga cagtggttca
     1621 aagttttttt cttccatttc aggtgtcgtg aggataatca ctttttttca ggttggaccg
     1681 gtgccaccat ggactataag gaccacgacg gagactacaa ggatcatgat attgattaca
     1741 aagacgatga cgataagatg gccccaaaga agaagcggaa ggtcggtatc cacggagtcc
     1801 cagcagccga caagaagtac agcatcggcc tggacatcgg caccaactct gtgggctggg
     1861 ccgtgatcac cgacgagtac aaggtgccca gcaagaaatt caaggtgctg ggcaacaccg
     1921 accggcacag catcaagaag aacctgatcg gagccctgct gttcgacagc ggcgaaacag
     1981 ccgaggccac ccggctgaag agaaccgcca gaagaagata caccagacgg aagaaccgga
     2041 tctgctatct gcaagagatc ttcagcaacg agatggccaa ggtggacgac agcttcttcc
     2101 acagactgga agagtccttc ctggtggaag aggataagaa gcacgagcgg caccccatct
     2161 tcggcaacat cgtggacgag gtggcctacc acgagaagta ccccaccatc taccacctga
     2221 gaaagaaact ggtggacagc accgacaagg ccgacctgcg gctgatctat ctggccctgg
     2281 cccacatgat caagttccgg ggccacttcc tgatcgaggg cgacctgaac cccgacaaca
     2341 gcgacgtgga caagctgttc atccagctgg tgcagaccta caaccagctg ttcgaggaaa
     2401 accccatcaa cgccagcggc gtggacgcca aggccatcct gtctgccaga ctgagcaaga
     2461 gcagacggct ggaaaatctg atcgcccagc tgcccggcga gaagaagaat ggcctgttcg
     2521 gaaacctgat tgccctgagc ctgggcctga cccccaactt caagagcaac ttcgacctgg
     2581 ccgaggatgc caaactgcag ctgagcaagg acacctacga cgacgacctg gacaacctgc
     2641 tggcccagat cggcgaccag tacgccgacc tgtttctggc cgccaagaac ctgtccgacg
     2701 ccatcctgct gagcgacatc ctgagagtga acaccgagat caccaaggcc cccctgagcg
     2761 cctctatgat caagagatac gacgagcacc accaggacct gaccctgctg aaagctctcg
     2821 tgcggcagca gctgcctgag aagtacaaag agattttctt cgaccagagc aagaacggct
     2881 acgccggcta cattgacggc ggagccagcc aggaagagtt ctacaagttc atcaagccca
     2941 tcctggaaaa gatggacggc accgaggaac tgctcgtgaa gctgaacaga gaggacctgc
     3001 tgcggaagca gcggaccttc gacaacggca gcatccccca ccagatccac ctgggagagc
     3061 tgcacgccat tctgcggcgg caggaagatt tttacccatt cctgaaggac aaccgggaaa
     3121 agatcgagaa gatcctgacc ttccgcatcc cctactacgt gggccctctg gccaggggaa
     3181 acagcagatt cgcctggatg accagaaaga gcgaggaaac catcaccccc tggaacttcg
     3241 aggaagtggt ggacaagggc gcttccgccc agagcttcat cgagcggatg accaacttcg
     3301 ataagaacct gcccaacgag aaggtgctgc ccaagcacag cctgctgtac gagtacttca
     3361 ccgtgtataa cgagctgacc aaagtgaaat acgtgaccga gggaatgaga aagcccgcct
     3421 tcctgagcgg cgagcagaaa aaggccatcg tggacctgct gttcaagacc aaccggaaag
     3481 tgaccgtgaa gcagctgaaa gaggactact tcaagaaaat cgagtgcttc gactccgtgg
     3541 aaatctccgg cgtggaagat cggttcaacg cctccctggg cacataccac gatctgctga
     3601 aaattatcaa ggacaaggac ttcctggaca atgaggaaaa cgaggacatt ctggaagata
     3661 tcgtgctgac cctgacactg tttgaggaca gagagatgat cgaggaacgg ctgaaaacct
     3721 atgcccacct gttcgacgac aaagtgatga agcagctgaa gcggcggaga tacaccggct
     3781 ggggcaggct gagccggaag ctgatcaacg gcatccggga caagcagtcc ggcaagacaa
     3841 tcctggattt cctgaagtcc gacggcttcg ccaacagaaa cttcatgcag ctgatccacg
     3901 acgacagcct gacctttaaa gaggacatcc agaaagccca ggtgtccggc cagggcgata
     3961 gcctgcacga gcacattgcc aatctggccg gcagccccgc cattaagaag ggcatcctgc
     4021 agacagtgaa ggtggtggac gagctcgtga aagtgatggg ccggcacaag cccgagaaca
     4081 tcgtgatcga aatggccaga gagaaccaga ccacccagaa gggacagaag aacagccgcg
     4141 agagaatgaa gcggatcgaa gagggcatca aagagctggg cagccagatc ctgaaagaac
     4201 accccgtgga aaacacccag ctgcagaacg agaagctgta cctgtactac ctgcagaatg
     4261 ggcgggatat gtacgtggac caggaactgg acatcaaccg gctgtccgac tacgatgtgg
     4321 accatatcgt gcctcagagc tttctggccg acgactccat cgacaacaag gtgctgacca
     4381 gaagcgacaa gaaccggggc aagagcgaca acgtgccctc cgaagaggtc gtgaagaaga
     4441 tgaagaacta ctggcggcag ctgctgaacg ccaagctgat tacccagaga aagttcgaca
     4501 atctgaccaa ggccgagaga ggcggcctga gcgaactgga taaggccggc ttcatcaaga
     4561 gacagctggt ggaaacccgg cagatcacaa agcacgtggc acagatcctg gactcccgga
     4621 tgaacactaa gtacgacgag aatgacaagc tgatccggga agtgaaagtg atcaccctga
     4681 agtccaagct ggtgtccgat ttccggaagg atttccagtt ttacaaagtg cgcgagatca
     4741 acaactacca ccacgcccac gacgcctacc tgaacgccgt cgtgggaacc gccctgatca
     4801 aaaagtaccc tgccctggaa agcgagttcg tgtacggcga ctacaaggtg tacgacgtgc
     4861 ggaagatgat cgccaagagc gagcaggaaa tcggcaaggc taccgccaag tacttcttct
     4921 acagcaacat catgaacttt ttcaagaccg agattaccct ggccaacggc gagatccgga
     4981 aggcccctct gatcgagaca aacggcgaaa ccggggagat cgtgtgggat aagggccggg
     5041 attttgccac cgtgcggaaa gtgctgagca tgccccaagt gaatatcgtg aaaaagaccg
     5101 aggtgcagac aggcggcttc agcaaagagt ctatcctgcc caagaggaac agcgataagc
     5161 tgatcgccag aaagaaggac tgggacccta agaagtacgg cggcttcgac agccccaccg
     5221 tggcctattc tgtgctggtg gtggccaaag tggaaaaggg caagtccaag aaactgaaga
     5281 gtgtgaaaga gctgctgggg atcaccatca tggaaagaag cagcttcgag aagaatccca
     5341 tcgactttct ggaagccaag ggctacaaag aagtgaaaaa ggacctgatc atcaagctgc
     5401 ctaagtactc cctgttcgag ctggaaaacg gccggaagag aatgctggcc tctgccggcg
     5461 aactgcagaa gggaaacgaa ctggccctgc cctccaaata tgtgaacttc ctgtacctgg
     5521 ccagccacta tgagaagctg aagggctccc ccgaggataa tgagcagaaa cagctgtttg
     5581 tggaacagca caagcactac ctggacgaga tcatcgagca gatcagcgag ttctccaaga
     5641 gagtgatcct ggccgacgct aatctggaca aagtgctgtc cgcctacaac aagcaccggg
     5701 ataagcccat cagagagcag gccgagaata tcatccacct gtttaccctg accaatctgg
     5761 gagcccctgc cgccttcaag tactttgaca ccaccatcga ccggaagagg tacaccagca
     5821 ccaaagaggt gctggacgcc accctgatcc accagagcat caccggcctg tacgagacac
     5881 ggatcgacct gtctcagctg ggaggcgaca gccccaagaa gaagagaaag gtggaggcca
     5941 gcggaagcgg agccactaac ttctccctgt tgaaacaagc aggggatgtc gaagagaatc
     6001 ccgggccaat ggtgtctaag ggcgaggagc tgatcaagga aaatatgcgt atgaaggtgg
     6061 tcatggaagg ttcggtcaac ggccaccaat tcaaatgcac aggtgaagga gaaggcaatc
     6121 cgtacatggg aactcaaacc atgaggatca aagtcatcga gggaggaccc ctgccatttg
     6181 cctttgacat tcttgccacg tcgttcatgt atggcagccg tacttttatc aagtacccga
     6241 aaggcattcc tgatttcttt aaacagtcct ttcctgaggg ttttacttgg gaaagagtta
     6301 cgagatacga agatggtgga gtcgtcaccg tcatgcagga caccagcctt gaggatggct
     6361 gtctcgttta ccacgtccaa gtcagagggg taaactttcc ctccaatggt cccgtgatgc
     6421 agaaaaagac caagggttgg gagcctaata cagagatgat gtatccagca gatggtggtc
     6481 tgaggggata cactcatatg gcactgaaag ttgatggtgg tggccatctg tcttgctctt
     6541 tcgtaacaac ttacaggtca aaaaagaccg tcgggaacat caagatgccc ggtatccatg
     6601 ccgttgatca ccgcctggaa aggttagagg aaagtgacaa tgaaatgttc gtagtacaac
     6661 gcgaacacgc agttgccaag ttcgccgggc ttggtggtgg gatggacgag ctgtacaagg
     6721 gaagcggagc cactaacttc tccctgttga aacaagcagg ggatgtcgaa gagaatcccg
     6781 ggccaatgac tgccatggag gagtcacagt cggatatcag cctcaagaga gcgctgccca
     6841 cctgcacaag cgcctctccc ccgcaaaaga aaaaaccact tgatggagag tatttcaccc
     6901 tcaagatccg cgggcgtaaa cgcttcgaga tgttccggga gctgaatgag gccttagagt
     6961 taaaggatgc ccatgctaca gaggagtctg gagacagcag ggctcactcc agctacctga
     7021 agaccaagaa gggccagtct acttcccgcc ataaaaaaac aatggtcaag aaagtggggc
     7081 ctgactcaga ctgataagaa ttcctagagc tcgctgatca gcctcgactg tgccttctag
     7141 ttgccagcca tctgttgttt gcccctcccc cgtgccttcc ttgaccctgg aaggtgccac
     7201 tcccactgtc ctttcctaat aaaatgagga aattgcatcg cattgtctga gtaggtgtca
     7261 ttctattctg gggggtgggg tggggcagga cagcaagggg gaggattggg aagagaatag
     7321 caggcatgct ggggagcggc cgcaggaacc cctagtgatg gagttggcca ctccctctct
     7381 gcgcgctcgc tcgctcactg aggccgggcg accaaaggtc gcccgacgcc cgggctttgc
     7441 ccgggcggcc tcagtgagcg agcgagcgcg cagctgcctg caggggcgcc tgatgcggta
     7501 ttttctcctt acgcatctgt gcggtatttc acaccgcata cgtcaaagca accatagtac
     7561 gcgccctgta gcggcgcatt aagcgcggcg ggtgtggtgg ttacgcgcag cgtgaccgct
     7621 acacttgcca gcgccctagc gcccgctcct ttcgctttct tcccttcctt tctcgccacg
     7681 ttcgccggct ttccccgtca agctctaaat cgggggctcc ctttagggtt ccgatttagt
     7741 gctttacggc acctcgaccc caaaaaactt gatttgggtg atggttcacg tagtgggcca
     7801 tcgccctgat agacggtttt tcgccctttg acgttggagt ccacgttctt taatagtgga
     7861 ctcttgttcc aaactggaac aacactcaac cctatctcgg gctattcttt tgatttataa
     7921 gggattttgc cgatttcggc ctattggtta aaaaatgagc tgatttaaca aaaatttaac
     7981 gcgaatttta acaaaattag acgtcaggtg gcacttttcg gggaaatgtg cgcggaaccc
     8041 ctatttgttt atttttctaa atacattcaa atatgtatcc gctcatgaga caataaccct
     8101 gataaatgct tcaataatat tgaaaaagga agagtatgag tattcaacat ttccgtgtcg
     8161 cccttattcc cttttttgcg gcattttgcc ttcctgtttt tgctcaccca gaaacgctgg
     8221 tgaaagtaaa agatgctgaa gatcagttgg gtgcacgagt gggttacatc gaactggatc
     8281 tcaacagcgg taagatcctt gagagttttc gccccgaaga acgttttcca atgatgagca
     8341 cttttaaagt tctgctatgt ggcgcggtat tatcccgtat tgacgccggg caagagcaac
     8401 tcggtcgccg catacactat tctcagaatg acttggttga gtactcacca gtcacagaaa
     8461 agcatcttac ggatggcatg acagtaagag aattatgcag tgctgccata accatgagtg
     8521 ataacactgc ggccaactta cttctgacaa cgatcggagg accgaaggag ctaaccgctt
     8581 ttttgcacaa catgggggat catgtaactc gccttgatcg ttgggaaccg gagctgaatg
     8641 aagccatacc aaacgacgag cgtgacacca cgatgcctgt agcaatggca acaacgttgc
     8701 gcaaactatt aactggcgaa ctacttactc tagcttcccg gcaacaatta atagactgga
     8761 tggaggcgga taaagttgca ggaccacttc tgcgctcggc ccttccggct ggctggttta
     8821 ttgctgataa atctggagcc ggtgagcgtg ggtctcgcgg tatcattgca gcactggggc
     8881 cagatggtaa gccctcccgt atcgtagtta tctacacgac ggggagtcag gcaactatgg
     8941 atgaacgaaa tagacagatc gctgagatag gtgcctcact gattaagcat tggtaactgt
     9001 cagaccaagt ttactcatat atactttaga ttgatttaaa acttcatttt taatttaaaa
     9061 ggatctaggt gaagatcctt tttgataatc tcatgaccaa aatcccttaa cgtgagtttt
     9121 cgttccactg agcgtcagac cccgtagaaa agatcaaagg atcttcttga gatccttttt
     9181 ttctgcgcgt aatctgctgc ttgcaaacaa aaaaaccacc gctaccagcg gtggtttgtt
     9241 tgccggatca agagctacca actctttttc cgaaggtaac tggcttcagc agagcgcaga
     9301 taccaaatac tgttcttcta gtgtagccgt agttaggcca ccacttcaag aactctgtag
     9361 caccgcctac atacctcgct ctgctaatcc tgttaccagt ggctgctgcc agtggcgata
     9421 agtcgtgtct taccgggttg gactcaagac gatagttacc ggataaggcg cagcggtcgg
     9481 gctgaacggg gggttcgtgc acacagccca gcttggagcg aacgacctac accgaactga
     9541 gatacctaca gcgtgagcta tgagaaagcg ccacgcttcc cgaagggaga aaggcggaca
     9601 ggtatccggt aagcggcagg gtcggaacag gagagcgcac gagggagctt ccagggggaa
     9661 acgcctggta tctttatagt cctgtcgggt ttcgccacct ctgacttgag cgtcgatttt
     9721 tgtgatgctc gtcagggggg cggagcctat ggaaaaacgc cagcaacgcg gcctttttac
     9781 ggttcctggc cttttgctgg ccttttgctc acatgctggg cccag
//
