LOCUS       sequence-396311-       15245 bp    DNA     circular SYN 15-DEC-2025
DEFINITION  Expresses the PE5max system to engineer the SF3B1 K700E mutation. 
            Co-expresses both a puromycin resistance gene and tagRFP for 
            selection..
ACCESSION   .
VERSION     .
KEYWORDS    .
SOURCE      synthetic DNA construct
  ORGANISM  synthetic DNA construct
REFERENCE   1  (bases 1 to 15245)
  AUTHORS   Fernandez M, Yu L, Jia Q, Wang X, Hart KL, Jia Z, Lin RJ, Wang L
  TITLE     Engineering oncogenic hotspot mutations on SF3B1 via CRISPR-directed
            PRECIS mutagenesis.
  JOURNAL   Cancer Res Commun. 2024 Aug 28. doi: 10.1158/2767-9764.CRC-24-0145.
  PUBMED    39194178
REFERENCE   2  (bases 1 to 15245)
  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: "Cancer Res 
            Commun. 2024 Aug 28. doi: 10.1158/2767-9764.CRC-24-0145."
COMMENT     Sequence Label: pCMV-PE5max-SF3B1-K700E
FEATURES             Location/Qualifiers
     source          1..15245
                     /mol_type="other DNA"
                     /organism="synthetic DNA construct"
     primer_bind     48..67
                     /label=pBR322ori-F
                     /note="pBR322 origin, forward primer"
     primer_bind     301..318
                     /label=L4440
                     /note="L4440 vector, forward primer"
     protein_bind    435..456
                     /label=CAP binding site
                     /bound_moiety="E. coli catabolite activator protein"
                     /note="CAP binding activates transcription in the presence 
                     of cAMP."
     promoter        join(471..476,477..494,495..501)
                     /label=lac promoter
                     /note="promoter for the E. coli lac operon"
     protein_bind    509..525
                     /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)."
     primer_bind     514..536
                     /label=M13/pUC Reverse
                     /note="In lacZ gene"
     primer_bind     533..549
                     /label=M13 rev
                     /note="common sequencing primer, one of multiple similar 
                     variants"
     primer_bind     533..549
                     /label=M13 Reverse
                     /note="In lacZ gene. Also called M13-rev"
     polyA_signal    620..844
                     /label=bGH poly(A) signal
                     /note="bovine growth hormone polyadenylation signal"
     primer_bind     833..850
                     /label=BGH-rev
                     /note="Bovine growth hormone terminator, reverse primer. 
                     Also called BGH reverse"
     CDS             complement(873..890)
                     /codon_start=1
                     /product="6xHis affinity tag"
                     /label=6xHis
                     /translation="HHHHHH"
     CDS             complement(3155..3211)
                     /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             complement(3221..3247)
                     /codon_start=1
                     /product="nuclear localization signal of human c-Myc 
                     proto-oncogene (Dang and Lee, 1988)"
                     /label=c-myc NLS
                     /translation="PAAKRVKLD"
     CDS             complement(3257..3277)
                     /codon_start=1
                     /product="nuclear localization signal of SV40 (simian virus
                     40) large T antigen"
                     /label=SV40 NLS
                     /translation="PKKKRKV"
     CDS             complement(5378..5398)
                     /codon_start=1
                     /product="nuclear localization signal of SV40 (simian virus
                     40) large T antigen"
                     /label=SV40 NLS
                     /translation="PKKKRKV"
     CDS             complement(5456..9556)
                     /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
                     RGHFLIEGDLNPDNSDVDKLFIQLVQTYNQLFEENPINASGVDAKAILSARLSKSRKLE
                     NLIAQLPGEKKNGLFGNLIALSLGLTPNFKSNFDLAEDAKLQLSKDTYDDDLDNLLAQI
                     GDQYADLFLAAKNLSDAILLSDILRVNTEITKAPLSASMIKRYDEHHQDLTLLKALVRQ
                     QLPEKYKEIFFDQSKNGYAGYIDGGASQEEFYKFIKPILEKMDGTEELLVKLKREDLLR
                     KQRTFDNGSIPHQIHLGELHAILRRQEDFYPFLKDNREKIEKILTFRIPYYVGPLARGN
                     SRFAWMTRKSEETITPWNFEEVVDKGASAQSFIERMTNFDKNLPNEKVLPKHSLLYEYF
                     TVYNELTKVKYVTEGMRKPAFLSGEQKKAIVDLLFKTNRKVTVKQLKEDYFKKIECFDS
                     VEISGVEDRFNASLGTYHDLLKIIKDKDFLDNEENEDILEDIVLTLTLFEDREMIEERL
                     KTYAHLFDDKVMKQLKRRRYTGWGRLSRKLINGIRDKQSGKTILDFLKSDGFANRNFMQ
                     LIHDDSLTFKEDIQKAQVSGQGDSLHEHIANLAGSPAIKKGILQTVKVVDELVKVMGRH
                     KPENIVIEMARENQTTQKGQKNSRERMKRIEEGIKELGSQILKEHPVENTQLQNEKLYL
                     YYLQNGRDMYVDQELDINRLSDYDVDAIVPQSFLKDDSIDNKVLTRSDKNRGKSDNVPS
                     EEVVKKMKNYWRQLLNAKLITQRKFDNLTKAERGGLSELDKAGFIKRQLVETRQITKHV
                     AQILDSRMNTKYDENDKLIREVKVITLKSKLVSDFRKDFQFYKVREINNYHHAHDAYLN
                     AVVGTALIKKYPKLESEFVYGDYKVYDVRKMIAKSEQEIGKATAKYFFYSNIMNFFKTE
                     ITLANGEIRKRPLIETNGETGEIVWDKGRDFATVRKVLSMPQVNIVKKTEVQTGGFSKE
                     SILPKRNSDKLIARKKDWDPKKYGGFDSPTVAYSVLVVAKVEKGKSKKLKSVKELLGIT
                     IMERSSFEKNPIDFLEAKGYKEVKKDLIIKLPKYSLFELENGRKRMLASAGELQKGNEL
                     ALPSKYVNFLYLASHYEKLKGSPEDNEQKQLFVEQHKHYLDEIIEQISEFSKRVILADA
                     NLDKVLSAYNKHRDKPIREQAENIIHLFTLTNLGAPAAFKYFDTTIDRKRYTSTKEVLD
                     ATLIHQSITGLYETRIDLSQLGGD"
     CDS             complement(9557..9577)
                     /codon_start=1
                     /product="nuclear localization signal of SV40 (simian virus
                     40) large T antigen"
                     /label=SV40 NLS
                     /translation="PKKKRKV"
     primer_bind     complement(9626..9645)
                     /label=T7
                     /note="T7 promoter, forward primer"
     promoter        complement(9627..9645)
                     /label=T7 promoter
                     /note="promoter for bacteriophage T7 RNA polymerase"
     promoter        complement(9687..9890)
                     /label=CMV promoter
                     /note="human cytomegalovirus (CMV) immediate early 
                     promoter"
     primer_bind     complement(9716..9736)
                     /label=CMV-F
                     /note="Human CMV immediate early promoter, forward primer"
     enhancer        9891..10270
                     /label=CMV enhancer
                     /note="human cytomegalovirus immediate early enhancer"
     primer_bind     10442..10461
                     /label=pRS-marker
                     /note="pRS vectors, use to sequence yeast selectable 
                     marker"
     misc_feature    10525..11113
                     /label=WPRE
                     /note="woodchuck hepatitis virus posttranscriptional 
                     regulatory element"
     CDS             10631..10642
                     /codon_start=1
                     /product="Factor Xa recognition and cleavage site"
                     /label=Factor Xa site
                     /translation="IEGR"
     primer_bind     11040..11060
                     /label=WPRE-R
                     /note="WPRE, reverse primer"
     CDS             complement(11129..11842)
                     /codon_start=1
                     /product="monomeric derivative of red fluorescent protein 
                     from Entacmaea quadricolor (Merzlyak et al., 2007)"
                     /label=TagRFP
                     /note="mammalian codon-optimized"
                     /translation="MVSKGEELIKENMHMKLYMEGTVNNHHFKCTSEGEGKPYEGTQTM
                     RIKVVEGGPLPFAFDILATSFMYGSRTFINHTQGIPDFFKQSFPEGFTWERVTTYEDGG
                     VLTATQDTSLQDGCLIYNVKIRGVNFPSNGPVMQKKTLGWEANTEMLYPADGGLEGRSD
                     MALKLVGGGHLICNFKTTYRSKKPAKNLKMPGVYYVDHRLERIKEADKETYVEQHEVAV
                     ARYCDLPSKLGHKLN"
     CDS             complement(11843..11899)
                     /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             complement(11915..12511)
                     /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(11995..12015)
                     /label=Puro-F
                     /note="Puromycin resistance gene, forward primer"
     primer_bind     12492..12511
                     /label=Puro-R
                     /note="Puromycin resistance gene, reverse primer. Also 
                     called puro-variant-R"
     promoter        complement(12537..12748)
                     /label=EF-1-alpha core promoter
                     /note="core promoter for human elongation factor 
                     EF-1-alpha"
     misc_RNA        complement(12857..12932)
                     /label=gRNA scaffold
                     /note="guide RNA scaffold for the Streptococcus pyogenes 
                     CRISPR/Cas9 system"
     promoter        complement(12954..13202)
                     /label=U6 promoter
                     /note="RNA polymerase III promoter for human U6 snRNA 
                     (Domitrovich & Kunkel, 2003)"
     misc_RNA        complement(13273..13348)
                     /label=gRNA scaffold
                     /note="guide RNA scaffold for the Streptococcus pyogenes 
                     CRISPR/Cas9 system"
     promoter        complement(13370..13618)
                     /label=U6 promoter
                     /note="RNA polymerase III promoter for human U6 snRNA 
                     (Domitrovich & Kunkel, 2003)"
     promoter        13668..13772
                     /gene="bla"
                     /label=AmpR promoter
     CDS             join(13773..13841,13842..14633)
                     /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(13991..14010)
                     /label=Amp-R
                     /note="Ampicillin resistance gene, reverse primer"
     rep_origin      join(14804..15245,1..147)
                     /direction=RIGHT
                     /label=ori
                     /note="high-copy-number ColE1/pMB1/pBR322/pUC origin of 
                     replication"
ORIGIN
        1 gtaagcggca gggtcggaac aggagagcgc acgagggagc ttccaggggg aaacgcctgg
       61 tatctttata gtcctgtcgg gtttcgccac ctctgacttg agcgtcgatt tttgtgatgc
      121 tcgtcagggg ggcggagcct atggaaaaac gccagcaacg cggccttttt acggttcctg
      181 gccttttgct ggccttttgc tcacatgttc tttcctgcgt tatcccctga ttctgtggat
      241 aaccgtatta ccgcctttga gtgagctgat accgctcgcc gcagccgaac gaccgagcgc
      301 agcgagtcag tgagcgagga agcggaagag cgcccaatac gcaaaccgcc tctccccgcg
      361 cgttggccga ttcattaatg cagctggcac gacaggtttc ccgactggaa agcgggcagt
      421 gagcgcaacg caattaatgt gagttagctc actcattagg caccctaggc tttacacttt
      481 atgcttccgg ctcgtatgtt gtgtggaatt gtgagcggat aacaatttca cacaggaaac
      541 agctatgacc atgattacgc caagctctag ctagaggtcg acggtatcga gccccagctg
      601 gttctttccg cctcagaagc catagagccc accgcatccc cagcatgcct gctattgtct
      661 tcccaatcct cccccttgct gtcctgcccc accccacccc ccagaataga atgacaccta
      721 ctcagacaat gcgatgcaat tttctcattt tattaggaaa ggacagtggg agtggcacct
      781 tccagggtca aggaaggcac gggggagggg caaacaacag atggctggca actagaaggc
      841 acagtcgagg ctgatcagcg ggtttaaact caatggtgat ggtgatgatg accggttaga
      901 aaaccttata aaggtcgggc aggttggcca gctgcaggat gttgccgtcc tcggtgaagt
      961 gcttaggagg gagaatgtgg ctccgcaggg ccttgtacac gatgtgttcc acggtccact
     1021 tccagctgtt ggggatgctt ccgggcactt cactctgctg gccagacaga gtgctctctt
     1081 cagagatgta ctgttttctg atgctataga acatggcgca ctccttgctc agagactcga
     1141 agcattcctt ttcctcgtcc cagttcacct ctgtagccag tctcaggatg aagatgggca
     1201 ggccttccag gggaggcacg tagttatcga tcagcagagg caggccgatc aggttgcctt
     1261 cctcgtcgat ttccagagaa aagtagtcgg ccagcatctc ggcctttttc ttcaggaact
     1321 ccacgatgta ttcagccagc ccctctttag gtccgtcctc ttctgtccag ccgctttctg
     1381 ggctatccag agccagcatg gccaggtcaa acagaggggc gggctcgctc aggcgtagca
     1441 cgccgaagtt ggcgaagtcg tagatgagga tctggtagaa cagctcttcg ctcagcttgg
     1501 tggtgttcag caggtacagc tttgtttgat gctgagccag ggcccattga gggttcacgc
     1561 agcccacgaa gctgtgattg tgcagcatct ctctcagcac ctcgtggccc tgctcgttga
     1621 tctcctcctg caggctcagc acggaggtca ggttgatgat tcttcttcta ggggtacaag
     1681 cggctgtcat ttcctttctg ctgtcgtcct ccaccatttc cacgtcgctg tcctcccggt
     1741 gccgttttct ggggttgctg gaggtagggc cccgcttctc gctcatttcg ctggtgccct
     1801 ttgtggtatc tccctccagg ctctggttct tggcggccac ctcggcagga gctggcagct
     1861 cgagcatttc ttcgtcctgc tgtctggcac ggccggagga gatatctgtc ttgtcctcgg
     1921 tcacgatggc ctgaggctga gagctcagag gtttgctcag aggttgcaga aaggcgtcca
     1981 gcttctgctc ccgagagtcg gtccgcacca tctggtgggc gtacacctta tcggaagaac
     2041 cgctggtgct ggatgaggtc agagaggttg tggacttaac catttcgcct gaggggccgg
     2101 ccaggccagg cagcagggtc tgtgtgaagt acattctgct gctgttagag cccagcagct
     2161 tgctctcaat gtgctgctgc actctttcca ggatgctttc ctcgtgcagg aagtgcacct
     2221 cgtgctttgt aggatgcaca ttgacgtcca cgttctgagg gctgatctcg aggctcaggt
     2281 acaggaaggg gtgggtgttc ttaggcaggt aggcggcgta cactgtctcg atggctttcc
     2341 gcaggctggt gctttccacc agtctgtggt tgatgaacag aagaaagatg cacttcttca
     2401 cagagtaatt ggcgttgctg atgtagccgt tcatcttaaa agccagggtc ttatcttcgc
     2461 agccgatctc gatcagttct ctggacacgg catttccgaa aatgcttctg atgttatcca
     2521 cggtgctggc atttggcagg gtgcgcacat cggccacggt ttctccctgc ttctttacgc
     2581 taaagctgat tccggcgttg tgcacggagt atctgccgac gacttccagg atcttgccgt
     2641 actcctcgct gggatttttc agggcctttc tccgggtggc gatgttgtag aacagatcct
     2701 ccactgttat ctgtgtaccc tggttgccgg cgcaaggctt tggcggggct ttcagcttgc
     2761 cgtcgctgta gctagcgcga taagcgcatt tgccgtcggc ggttttggtg gtgatggtca
     2821 catgggccac gtggctgatg ctggccaggg cctcgcctct gaagccgtag gtactgatag
     2881 aggccagatc ctcaaaagac tgcagcttag atgtggtgaa ccgttcacac acgatgtcca
     2941 gatcttcctt tctgatgcct gtgccgttgt cttggatctg gatcagcttc agtccgccct
     3001 ccttcacgat cacctgaatg cttgtggact tggcgtccag acagttctcg atcatctcct
     3061 ttattgcatt ggcaggtctc tggatcacct cgccggcggc aattctgttc accactgtct
     3121 cgtccagtct ccggatgact ccagcaacga agctaggtcc agggttctcc tccacgtctc
     3181 cagcctgctt cagcaggctg aagttagtag ctccgcttcc gtccagcttc actctcttag
     3241 cggcagggcc agagccgact ttcctcttct tcttgggaga ctcgaattcg ctgccgtcgg
     3301 cggttctttt tgagccgcca gaaggggagg agttttcaat cagcagagtg gaagtgtcgg
     3361 gagtttcagt aatagcagcc tttctggcgg cctggtcggc catccgattg ccccttgcct
     3421 cggcggagtg tcccttctgg tgtccaggac agtggatgat gctcagccgc ttgggcagaa
     3481 acagggcctt cagcagggcc aggatctcat ccttgttctt gatctccttg ccctcagagg
     3541 tcagccagcc ccggcgcctg tagatctctc cgtggatgtg tgcggtggcg aaggcatatc
     3601 tggagtctgt gtacacgttc agcttcttgc cctcggccat cttcagggcc tgggtcaggg
     3661 cgatcagctc ggcccgctgg gcggatgtgc ctgctggcag ggccttggcc cagatcactt
     3721 ctgtctctgt ggtcactgct gctcctgcct tcctctggcc ctcctgcagc agggagcttc
     3781 catcggtata ccatgtgtga tcggcgtcag gcaggggctg atcggtcagg tctggccttg
     3841 ttccgtgtgc ctctgccagg atgtccagac agttgtgctg caggccctcc tctggcagag
     3901 gcagcagggt ggctggattc agggccacca cagggccaaa ctgcacgcga tcggtgtcca
     3961 gcagcagggc ctggtagtgt gtcatccggg cgttagacag ccagcgatct ggaggctgct
     4021 tcaccagggc ctccactgcg tgtggtgcca ggatgaccag tggctgtccc atggtcagct
     4081 tgccggcatc ctttgtcagc acggcgattg ctgccaccat cctcaggcat ggtggccatc
     4141 ctgctgccac tgggtccagc ttcttggaca gataggccac gggccgtctc catggtccca
     4201 gcttctgggt cagcacgccc ttggcgtagc cctgcttctc atccacaaac agctcgaaag
     4261 gcttggtcag gtctggcagg cccagggctg gtgctgtcag cagggcctgc ttgatctcct
     4321 gataggcctt ctgctggtca gggccccaat taaacagggt gcccggcttg gtcagtgggt
     4381 acagaggtgc tgccatctct gcgaatcctg ggatgaacag cctgcaaaat cctgctttgc
     4441 ccaggaactc cctcagctgt cttggggtct tgggtgttgg ctggcccatc actgtctcct
     4501 tcctggcctc tgtcagccat ctctggccct ccttcagcag atagcccagg tacttcacct
     4561 gcttctgaca gatctgggcc ttcttggcgg atgccctata tcccaggttt cccagggtct
     4621 gcagcagggc gcgtgttccc tgctggcaat ccagctcgct ggtggcggcc agcagcagat
     4681 cgtccacgta ctgcagcagg atcagatctg ggtgctggat ccggaaatct gccaggtcgc
     4741 ggtgcagggc ctcgttaaac agtgttgggc tattcttgaa gccctgaggc aggcgtgtcc
     4801 aggtcagctg gccagagatg cccatctcag ggtccctcca ctcaaaggcg aacagtggct
     4861 ggctggtagg gtgcagtctc agacaaaaga aggcatcctt caggtccagc actgtatacc
     4921 actggtgaga agggggcagg ccggacagca gattgtaagg gttgggcacg gtggggtgga
     4981 tatcctccac cctcttgttc acctctctca gatcctgcac gggtctatag tcattggtgc
     5041 ctggcttctt cacgggcagc agtggtgtgt tccaggggct ctggcatggc accaggatgc
     5101 cctggtccag cagcctctgg atgtgaggct tgatgcccag tctggcctcc tgagacatag
     5161 ggtactgctt gatgctcacg ggtgtagagg tggccttcag agggatgatc aggggtgcct
     5221 gccgcacggc caggcccatt cctcctgtct cggcccaggc ctggggaaag tcggacagcc
     5281 aggtggagcc cagggacacg tcgggttcct tgcttgtttc atgcagtcta tactcgtctt
     5341 caatgttcag ggtgcttccg ccgctagagc ctccgctcac ctttcttttc ttcttagggc
     5401 tctcgaattc agagccgtcg gcggtccgct tgctgccacc agagcttccg ccggagtcac
     5461 ctcccagctg agacaggtcg atccgtgtct cgtacaggcc ggtgatgctc tggtggatca
     5521 gggtggcgtc cagcacctct ttggtgctgg tgtacctctt ccggtcgatg gtggtgtcaa
     5581 agtacttgaa ggcggcaggg gctcccagat tggtcagggt aaacaggtgg atgatattct
     5641 cggcctgctc tctgatgggc ttatcccggt gcttgttgta ggcggacagc actttgtcca
     5701 gattagcgtc ggccaggatc actctcttgg agaactcgct gatctgctcg atgatctcgt
     5761 ccaggtagtg cttgtgctgt tccacaaaca gctgtttctg ctcattatcc tcgggggagc
     5821 ccttcagctt ctcatagtgg ctggccaggt acaggaagtt cacatatttg gagggcaggg
     5881 ccagttcgtt tcccttctgc agttcgccgg cagaggccag cattctcttc cggccgtttt
     5941 ccagctcgaa cagggagtac ttaggcagct tgatgatcag gtcctttttc acttctttgt
     6001 agcccttggc ttccagaaag tcgatgggat tcttctcgaa gctgcttctt tccatgatgg
     6061 tgatccccag cagctctttc acactcttca gtttcttgga cttgcccttt tccactttgg
     6121 ccaccaccag cacagaatag gccacggtgg ggctgtcgaa gccgccgtac ttcttagggt
     6181 cccagtcctt ctttctggcg atcagcttat cgctgttcct cttgggcagg atagactctt
     6241 tgctgaagcc gcctgtctgc acctcggtct ttttcacgat attcacttgg ggcatgctca
     6301 gcactttccg cacggtggca aaatcccggc ccttatccca cacgatctcc ccggtttcgc
     6361 cgtttgtctc gatcagaggc cgcttccgga tctcgccgtt ggccagggta atctcggtct
     6421 tgaaaaagtt catgatgttg ctgtagaaga agtacttggc ggtagccttg ccgatttcct
     6481 gctcgctctt ggcgatcatc ttccgcacgt cgtacacctt gtagtcgccg tacacgaact
     6541 cgctttccag cttagggtac tttttgatca gggcggttcc cacgacggcg ttcaggtagg
     6601 cgtcgtgggc gtggtggtag ttgttgatct cgcgcacttt gtaaaactgg aaatccttcc
     6661 ggaaatcgga caccagcttg gacttcaggg tgatcacttt cacttcccgg atcagcttgt
     6721 cattctcgtc gtacttagtg ttcatccggg agtccaggat ctgtgccacg tgctttgtga
     6781 tctgccgggt ttccaccagc tgtctcttga tgaagccggc cttatccagt tcgctcaggc
     6841 cgcctctctc ggccttggtc agattgtcga actttctctg ggtaatcagc ttggcgttca
     6901 gcagctgccg ccagtagttc ttcatcttct tcacgacctc ttcggagggc acgttgtcgc
     6961 tcttgccccg gttcttgtcg cttctggtca gcaccttgtt gtcgatggag tcgtccttca
     7021 gaaagctctg aggcacgata gcgtccacat cgtagtcgga cagccggttg atgtccagtt
     7081 cctggtccac gtacatatcc cgcccattct gcaggtagta caggtacagc ttctcgttct
     7141 gcagctgggt gttttccacg gggtgttctt tcaggatctg gctgcccagc tctttgatgc
     7201 cctcttcgat ccgcttcatt ctctcgcggc tgttcttctg tcccttctgg gtggtctggt
     7261 tctctctggc catttcgatc acgatgttct cgggcttgtg ccggcccatc actttcacga
     7321 gctcgtccac caccttcact gtctgcagga tgcccttctt aatggcgggg ctgccggcca
     7381 gattggcaat gtgctcgtgc aggctatcgc cctggccgga cacctgggct ttctggatgt
     7441 cctctttaaa ggtcaggctg tcgtcgtgga tcagctgcat gaagtttctg ttggcgaagc
     7501 cgtcggactt caggaaatcc aggattgtct tgccggactg cttgtcccgg atgccgttga
     7561 tcagcttccg gctcagcctg ccccagccgg tgtatctccg ccgcttcagc tgcttcatca
     7621 ctttgtcgtc gaacaggtgg gcataggttt tcagccgttc ctcgatcatc tctctgtcct
     7681 caaacagtgt cagggtcagc acgatatctt ccagaatgtc ctcgttttcc tcattgtcca
     7741 ggaagtcctt gtccttgata attttcagca gatcgtggta tgtgcccagg gaggcgttga
     7801 accgatcttc cacgccggag atttccacgg agtcgaagca ctcgattttc ttgaagtagt
     7861 cctctttcag ctgcttcacg gtcactttcc ggttggtctt gaacagcagg tccacgatgg
     7921 cctttttctg ctcgccgctc aggaaggcgg gctttctcat tccctcggtc acgtatttca
     7981 ctttggtcag ctcgttatac acggtgaagt actcgtacag caggctgtgc ttgggcagca
     8041 ccttctcgtt gggcaggttc ttatcgaagt tggtcatccg ctcgatgaag ctctgggcgg
     8101 aagcgccctt gtccaccact tcctcgaagt tccagggggt gatggtttcc tcgctctttc
     8161 tggtcatcca ggcgaatctg ctgtttcccc tggccagagg gcccacgtag taggggatgc
     8221 ggaaggtcag gatcttctcg atcttttccc ggttgtcctt caggaatggg taaaaatctt
     8281 cctgccgccg cagaatggcg tgcagctctc ccaggtggat ctggtggggg atgctgccgt
     8341 tgtcgaaggt ccgctgcttc cgcagcaggt cctctctctt cagcttcacg agcagttcct
     8401 cggtgccgtc catcttttcc aggatgggct tgatgaactt gtagaactct tcctggctgg
     8461 ctccgccgtc aatgtagccg gcgtagccgt tcttgctctg gtcgaagaaa atctctttgt
     8521 acttctcagg cagctgctgc cgcacgagag ctttcagcag ggtcaggtcc tggtggtgct
     8581 cgtcgtatct cttgatcata gaggcgctca ggggggcctt ggtgatctcg gtgttcactc
     8641 tcaggatgtc gctcagcagg atggcgtcgg acaggttctt ggcggccaga aacaggtcgg
     8701 cgtactggtc gccgatctgg gccagcaggt tgtccaggtc gtcgtcgtag gtgtccttgc
     8761 tcagctgcag tttggcatcc tcggccaggt cgaagttgct cttgaagttg ggggtcaggc
     8821 ccaggctcag ggcaatcagg tttccgaaca ggccattctt cttctcgccg ggcagctggg
     8881 cgatcagatt ttccagcttt ctgctcttgc tcagtctggc agacaggatg gccttggcgt
     8941 ccacgccgct ggcgttgatg gggttttcct cgaacagctg gttgtaggtc tgcaccagct
     9001 ggatgaacag cttgtccacg tcgctgttgt cggggttcag gtcgccctcg atcaggaagt
     9061 ggccccggaa cttgatcatg tgggccaggg ccagatagat cagccgcagg tcggccttgt
     9121 cggtgctgtc caccagtttc tttctcaggt ggtagatggt ggggtacttc tcgtggtagg
     9181 ccacctcgtc cacgatgttg ccgaagatgg ggtgccgctc gtgcttctta tcctcttcca
     9241 ccaggaagga ctcttccagt ctgtggaaga agctgtcgtc caccttggcc atctcgttgc
     9301 tgaagatctc ttgcagatag cagatccggt tcttccgtct ggtgtatctt cttctggcgg
     9361 ttctcttcag ccgggtggcc tcggctgttt cgccgctgtc gaacagcagg gctccgatca
     9421 ggttcttctt gatgctgtgc cggtcggtgt tgcccagcac cttgaatttc ttgctgggca
     9481 ccttgtactc gtcggtgatc acggcccagc ccacagagtt ggtgccgatg tccaggccga
     9541 tgctgtactt cttgtcgact ttccgcttct tctttggtga ctcgaactcg cttccgtcgg
     9601 ctgtccgttt catggtggcg gctctcccta tagtgagtcg tattagcggc cgcggatctc
     9661 tagcggatct gacggttcac taaaccagct ctgcttatat agacctccca ccgtacacgc
     9721 ctaccgccca tttgcgtcaa tggggcggag ttgttacgac attttggaaa gtcccgttga
     9781 ttttggtgcc aaaacaaact cccattgacg tcaatggggt ggagacttgg aaatccccgt
     9841 gagtcaaacc gctatccacg cccattgatg tactgccaaa accgcatcac catggtaata
     9901 gcgatgacta atacgtagat gtactgccaa gtaggaaagt cccataaggt catgtactgg
     9961 gcataatgcc aggcgggcca tttaccgtca ttgacgtcaa tagggggcgt acttggcata
    10021 tgatacactt gatgtactgc caagtgggca gtttaccgta aatactccac ccattgacgt
    10081 caatggaaag tccctattgg cgttactatg ggaacatacg tcattattga cgtcaatggg
    10141 cgggggtcgt tgggcggtca gccaggcggg ccatttaccg taagttatgt aacgcggaac
    10201 tccatatatg ggctatgaac taatgacccc gtaattgatt actattaata actagtcaat
    10261 aatcaatgtc aacgcgtata tctggcccgt acatcgcgaa gcagcgcaaa acgcctaacc
    10321 ctaagcagat tcttcatgca attgtcggtc aagccttgcc ttgttgtagc ttaaattttg
    10381 ctcgcgcact actcagcgac ctccaacaca caagcaggga gcagatactg gcttaactat
    10441 gcggcatcag agcagattgt actgagagta gaccctaggg gatcgggaga tcgatctccc
    10501 gatccgtcga cgcttaaagt cgacgcgggg aggcggccca aagggagatc cgactcgtct
    10561 gagggcgaag gcgaagacgc ggaagaggcc gcagagccgg cagcaggccg cgggaaggaa
    10621 ggtccgctgg attgagggcc gaagggacgt agcagaagga cgtcccgcgc agaatccagg
    10681 tggcaacaca ggcgagcagc caaggaaagg acgatgattt ccccgacaac accacggaat
    10741 tgtcagtgcc caacagccga gcccctgtcc agcagcgggc aaggcaggcg gcgatgagtt
    10801 ccgccgtggc aatagggagg gggaaagcga aagtcccgga aaggagctga caggtggtgg
    10861 caatgcccca accagtgggg gttgcgtcag caaacacagt gcacaccacg ccacgttgcc
    10921 tgacaacggg ccacaactcc tcataaagag acagcaacca ggatttatac aaggaggaga
    10981 aaatgaaagc catacgggaa gcaatagcat gatacaaagg cattaaagca gcgtatccac
    11041 atagcgtaaa aggagcaaca tagttaagaa taccagtcaa tctttcacaa attttgtaat
    11101 ccagaggttg attgtcgact taacgcgttc aattaagttt gtgccccagt ttgctaggga
    11161 ggtcgcagta tctggccaca gccacctcgt gctgctcgac gtaggtctct ttgtcggcct
    11221 ccttgattct ttccagtctg tggtccacat agtagacgcc gggcatcttg aggttcttag
    11281 cgggtttctt ggatctgtat gtggtcttga agttgcagat caggtggccc ccgcccacga
    11341 gcttcagggc catgtcgctt ctgccttcca ggccgccgtc agcggggtac agcatctcgg
    11401 tgttggcctc ccagccgagt gttttcttct gcatcacagg gccgttggat gggaagttca
    11461 cccctctgat cttgacgttg tagatgaggc agccgtcctg gaggctggtg tcctgggtag
    11521 cggtcagcac gcccccgtct tcgtatgtgg tgactctctc ccatgtgaag ccctcaggga
    11581 aggactgctt aaagaagtcg gggatgccct gggtgtggtt gatgaaggtt ctgctgccgt
    11641 acatgaagct ggtagccagg atgtcgaagg cgaaggggag agggccgccc tcgaccacct
    11701 tgattctcat ggtctgggtg ccctcgtagg gcttgccttc gccctcggat gtgcacttga
    11761 agtggtggtt gttcacggtg ccctccatat acagcttcat gtgcatgttc tccttaatca
    11821 gctcttcgcc cttagacacc atggggccgg ggttctcctc cacgtcgccg gcctgcttca
    11881 gcaggctgaa gttggtggcg ccgctgccgc tagcggcacc gggcttgcgg gtcatgcacc
    11941 aggtgcgcgg tccttcgggc acctcgacgt cggcggtgac ggtgaagccg agccgctcgt
    12001 agaaggggag gttgcggggc gcggaggtct ccaggaaggc gggcaccccg gcgcgctcgg
    12061 ccgcctccac tccggggagc acgacggcgc tgcccagacc cttgccctgg tggtcgggcg
    12121 agactccgac ggtggccagg aaccacgcgg gctccttggg ccggtgcggc gccaggaggc
    12181 cttccatctg ttgctgcgcg gccagccggg aaccgctcaa ctcggccatg cgcgggccga
    12241 tctcggcgaa caccgccccc gcttcgacgc tctccggcgt ggtccagacc gccacggcgg
    12301 cgccgtcgtc cgcgacccac accttgccga tgtcgagccc gacgcgcgtg aggaagagtt
    12361 cttgcagctc ggtgacccgc tcgatgtggc ggtccggatc gacggtgtgg cgcgtggcgg
    12421 ggtagtcggc gaacgcggcg gcgagggtgc gtacggccct ggggacgtcg tcgcgggtgg
    12481 cgaggcgcac cgtgggcttg tactcggtca tggtggcgga tcccgcgtca cgacacctgt
    12541 gttctggcgg caaacccgtt gcgaaaaaga acgttcacgg cgactactgc acttatatac
    12601 ggttctcccc caccctcggg aaaaaggcgg agccagtaca cgacatcact ttcccagttt
    12661 accccgcgcc accttctcta ggcaccggtt caattgccga cccctccccc caacttctcg
    12721 gggactgtgg gcgatgtgcg ctctgcccac tgacgggcac cggagccact cgagcggccc
    12781 aagcttaaaa aaagttgctg cttcagccaa ggcagcaatg gccaaagcac tgatggtccg
    12841 aacttcctgc tgctcagcac cgactcggtg ccactttttc aagttgataa cggactagcc
    12901 ttattttaac ttgctatttc tagctctaaa actggctgaa gcagcaactc ctcggtgttt
    12961 cgtcctttcc acaagatata taaagccaag aaatcgaaat actttcaagt tacggtaagc
    13021 atatgatagt ccattttaaa acataatttt aaaactgcaa actacccaag aaattattac
    13081 tttctacgtc acgtattttg tactaatatc tttgtgttta cagtcaaatt aattctaatt
    13141 atctctctaa cagccttgta tcgtatatgc aaatatgaag gaatcatggg aaataggccc
    13201 tcttcctgcc cgaccttggt accggatcca gtcgactgaa ttggttcctt taaagcctgc
    13261 gaattcaaaa aagcaccgac tcggtgccac tttttcaagt tgataacgga ctagccttat
    13321 tttaacttgc tatttctagc tctaaaacaa cttcctgctg ctcatccacg gtgtttcgtc
    13381 ctttccacaa gatatataaa gccaagaaat cgaaatactt tcaagttacg gtaagcatat
    13441 gatagtccat tttaaaacat aattttaaaa ctgcaaacta cccaagaaat tattactttc
    13501 tacgtcacgt attttgtact aatatctttg tgtttacagt caaattaatt ctaattatct
    13561 ctctaacagc cttgtatcgt atatgcaaat atgaaggaat catgggaaat aggccctctc
    13621 tgggtcccct cgcgtcgacg tcaggtggca cttttcgggg aaatgtgcgc ggaaccccta
    13681 tttgtttatt tttctaaata cattcaaata tgtatccgct catgagacaa taaccctgat
    13741 aaatgcttca ataatattga aaaaggaaga gtatgagtat tcaacatttc cgtgtcgccc
    13801 ttattccctt ttttgcggca ttttgccttc ctgtttttgc tcacccagaa acgctggtga
    13861 aagtaaaaga tgctgaagat cagttgggtg cacgagtggg ttacatcgaa ctggatctca
    13921 acagcggtaa gatccttgag agttttcgcc ccgaagaacg ttttccaatg atgagcactt
    13981 ttaaagttct gctatgtggc gcggtattat cccgtattga cgccgggcaa gagcaactcg
    14041 gtcgccgcat acactattct cagaatgact tggttgagta ctcaccagtc acagaaaagc
    14101 atcttacgga tggcatgaca gtaagagaat tatgcagtgc tgccataacc atgagtgata
    14161 acactgcggc caacttactt ctgacaacga tcggaggacc gaaggagcta accgcttttt
    14221 tgcacaacat gggggatcat gtaactcgcc ttgatcgttg ggaaccggag ctgaatgaag
    14281 ccataccaaa cgacgagcgt gacaccacga tgcctgtagc aatggcaaca acgttgcgca
    14341 aactattaac tggcgaacta cttactctag cttcccggca acaattaata gactggatgg
    14401 aggcggataa agttgcagga ccacttctgc gctcggccct tccggctggc tggtttattg
    14461 ctgataaatc tggagccggt gagcgtgggt ctcgcggtat cattgcagca ctggggccag
    14521 atggtaagcc ctcccgtatc gtagttatct acacgacggg gagtcaggca actatggatg
    14581 aacgaaatag acagatcgct gagataggtg cctcactgat taagcattgg taactgtcag
    14641 accaagttta ctcatatata ctttagattg atttaaaact tcatttttaa tttaaaagga
    14701 tctaggtgaa gatccttttt gataatctca tgaccaaaat cccttaacgt gagttttcgt
    14761 tccactgagt gtcagacccc gtagaaaaga tcaaaggatc ttcttgagat cctttttttc
    14821 tgcgcgtaat ctgctgcttg caaacaaaaa aaccaccgct accagcggtg gtttgtttgc
    14881 cggatcaaga gctaccaact ctttttccga aggtaactgg cttcagcaga gcgcagatac
    14941 caaatactgt tcttctagtg tagccgtagt taggccacca cttcaagaac tctgtagcac
    15001 cgcctacata cctcgctctg ctaatcctgt taccagtggc tgctgccagt ggcgataagt
    15061 cgtgtcttac cgggttggac tcaagacgat agttaccgga taaggcgcag cggtcgggct
    15121 gaacgggggg ttcgtgcaca cagcccagct tggagcgaac gacctacacc gaactgagat
    15181 acctacagcg tgagctatga gaaagcgcca cgcttcccga agggagaaag gcggacaggt
    15241 atccg
//
