LOCUS       sequence-421515-       12649 bp    DNA     circular SYN 15-DEC-2025
DEFINITION  EFS-Cas9-P2A-Puro.
ACCESSION   .
VERSION     .
KEYWORDS    .
SOURCE      synthetic DNA construct
  ORGANISM  synthetic DNA construct
REFERENCE   1  (bases 1 to 12649)
  AUTHORS   Tan IL, Perez AR, Lew RJ, Sun X, Baldwin A, Zhu YK, Shah MM, Berger 
            MS, Doudna JA, Fellmann C
  TITLE     Targeting the non-coding genome and temozolomide signature enables 
            CRISPR-mediated glioma oncolysis.
  JOURNAL   Cell Rep. 2023 Nov 28;42(11):113339. doi: 
            10.1016/j.celrep.2023.113339. Epub 2023 Nov 2.
  PUBMED    37917583
REFERENCE   2  (bases 1 to 12649)
  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: "Cell Rep.";
            date: "2023-11-28"; volume: "42(11):113339. doi"; pages: " 
            10.1016/j.celrep.2023.113339. Epub 2023 Nov 2"
COMMENT     Sequence Label: pCF226_EFS-Cas9-P2A-Puro
FEATURES             Location/Qualifiers
     source          1..12649
                     /mol_type="other DNA"
                     /organism="synthetic DNA construct"
     primer_bind     complement(47..66)
                     /label=pRS-marker
                     /note="pRS vectors, use to sequence yeast selectable 
                     marker"
     enhancer        238..617
                     /label=CMV enhancer
                     /note="human cytomegalovirus immediate early enhancer"
     promoter        619..817
                     /label=CMV promoter
                     /note="human cytomegalovirus (CMV) immediate early 
                     promoter"
     primer_bind     772..792
                     /label=CMV-F
                     /note="Human CMV immediate early promoter, forward primer"
     LTR             835..1015
                     /label=5' LTR (truncated)
                     /note="truncated 5' long terminal repeat (LTR) from HIV-1"
     misc_feature    1062..1187
                     /label=HIV-1 Psi
                     /note="packaging signal of human immunodeficiency virus 
                     type 1"
     misc_feature    1680..1913
                     /label=RRE
                     /note="The Rev response element (RRE) of HIV-1 allows for 
                     Rev-dependent mRNA export from the nucleus to the 
                     cytoplasm."
     CDS             2098..2142
                     /codon_start=1
                     /product="antigenic peptide corresponding to amino acids 
                     655 to 669 of the HIV envelope protein gp41 (Lutje Hulsik 
                     et al., 2013)"
                     /label=gp41 peptide
                     /note="recognized by the 2H10 single-chain llama nanobody"
                     /translation="KNEQELLELDKWASL"
     misc_feature    2440..2557
                     /label=cPPT/CTS
                     /note="central polypurine tract and central termination 
                     sequence of HIV-1"
     promoter        2651..2862
                     /label=EF-1-alpha core promoter
                     /note="core promoter for human elongation factor 
                     EF-1-alpha"
     regulatory      2881..2890
                     /label=Kozak sequence
                     /note="vertebrate consensus sequence for strong initiation 
                     of translation (Kozak, 1987)"
                     /regulatory_class="other"
     CDS             2887..6990
                     /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="MDKKYSIGLDIGTNSVGWAVITDEYKVPSKKFKVLGNTDRHSIKK
                     NLIGALLFDSGETAEATRLKRTARRRYTRRKNRICYLQEIFSNEMAKVDDSFFHRLEES
                     FLVEEDKKHERHPIFGNIVDEVAYHEKYPTIYHLRKKLVDSTDKADLRLIYLALAHMIK
                     FRGHFLIEGDLNPDNSDVDKLFIQLVQTYNQLFEENPINASGVDAKAILSARLSKSRRL
                     ENLIAQLPGEKKNGLFGNLIALSLGLTPNFKSNFDLAEDAKLQLSKDTYDDDLDNLLAQ
                     IGDQYADLFLAAKNLSDAILLSDILRVNTEITKAPLSASMIKRYDEHHQDLTLLKALVR
                     QQLPEKYKEIFFDQSKNGYAGYIDGGASQEEFYKFIKPILEKMDGTEELLVKLNREDLL
                     RKQRTFDNGSIPHQIHLGELHAILRRQEDFYPFLKDNREKIEKILTFRIPYYVGPLARG
                     NSRFAWMTRKSEETITPWNFEEVVDKGASAQSFIERMTNFDKNLPNEKVLPKHSLLYEY
                     FTVYNELTKVKYVTEGMRKPAFLSGEQKKAIVDLLFKTNRKVTVKQLKEDYFKKIECFD
                     SVEISGVEDRFNASLGTYHDLLKIIKDKDFLDNEENEDILEDIVLTLTLFEDREMIEER
                     LKTYAHLFDDKVMKQLKRRRYTGWGRLSRKLINGIRDKQSGKTILDFLKSDGFANRNFM
                     QLIHDDSLTFKEDIQKAQVSGQGDSLHEHIANLAGSPAIKKGILQTVKVVDELVKVMGR
                     HKPENIVIEMARENQTTQKGQKNSRERMKRIEEGIKELGSQILKEHPVENTQLQNEKLY
                     LYYLQNGRDMYVDQELDINRLSDYDVDHIVPQSFLKDDSIDNKVLTRSDKNRGKSDNVP
                     SEEVVKKMKNYWRQLLNAKLITQRKFDNLTKAERGGLSELDKAGFIKRQLVETRQITKH
                     VAQILDSRMNTKYDENDKLIREVKVITLKSKLVSDFRKDFQFYKVREINNYHHAHDAYL
                     NAVVGTALIKKYPKLESEFVYGDYKVYDVRKMIAKSEQEIGKATAKYFFYSNIMNFFKT
                     EITLANGEIRKRPLIETNGETGEIVWDKGRDFATVRKVLSMPQVNIVKKTEVQTGGFSK
                     ESILPKRNSDKLIARKKDWDPKKYGGFDSPTVAYSVLVVAKVEKGKSKKLKSVKELLGI
                     TIMERSSFEKNPIDFLEAKGYKEVKKDLIIKLPKYSLFELENGRKRMLASAGELQKGNE
                     LALPSKYVNFLYLASHYEKLKGSPEDNEQKQLFVEQHKHYLDEIIEQISEFSKRVILAD
                     ANLDKVLSAYNKHRDKPIREQAENIIHLFTLTNLGAPAAFKYFDTTIDRKRYTSTKEVL
                     DATLIHQSITGLYETRIDLSQLGGD"
     CDS             2887..6990
                     /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="MDKKYSIGLDIGTNSVGWAVITDEYKVPSKKFKVLGNTDRHSIKK
                     NLIGALLFDSGETAEATRLKRTARRRYTRRKNRICYLQEIFSNEMAKVDDSFFHRLEES
                     FLVEEDKKHERHPIFGNIVDEVAYHEKYPTIYHLRKKLVDSTDKADLRLIYLALAHMIK
                     FRGHFLIEGDLNPDNSDVDKLFIQLVQTYNQLFEENPINASGVDAKAILSARLSKSRRL
                     ENLIAQLPGEKKNGLFGNLIALSLGLTPNFKSNFDLAEDAKLQLSKDTYDDDLDNLLAQ
                     IGDQYADLFLAAKNLSDAILLSDILRVNTEITKAPLSASMIKRYDEHHQDLTLLKALVR
                     QQLPEKYKEIFFDQSKNGYAGYIDGGASQEEFYKFIKPILEKMDGTEELLVKLNREDLL
                     RKQRTFDNGSIPHQIHLGELHAILRRQEDFYPFLKDNREKIEKILTFRIPYYVGPLARG
                     NSRFAWMTRKSEETITPWNFEEVVDKGASAQSFIERMTNFDKNLPNEKVLPKHSLLYEY
                     FTVYNELTKVKYVTEGMRKPAFLSGEQKKAIVDLLFKTNRKVTVKQLKEDYFKKIECFD
                     SVEISGVEDRFNASLGTYHDLLKIIKDKDFLDNEENEDILEDIVLTLTLFEDREMIEER
                     LKTYAHLFDDKVMKQLKRRRYTGWGRLSRKLINGIRDKQSGKTILDFLKSDGFANRNFM
                     QLIHDDSLTFKEDIQKAQVSGQGDSLHEHIANLAGSPAIKKGILQTVKVVDELVKVMGR
                     HKPENIVIEMARENQTTQKGQKNSRERMKRIEEGIKELGSQILKEHPVENTQLQNEKLY
                     LYYLQNGRDMYVDQELDINRLSDYDVDHIVPQSFLKDDSIDNKVLTRSDKNRGKSDNVP
                     SEEVVKKMKNYWRQLLNAKLITQRKFDNLTKAERGGLSELDKAGFIKRQLVETRQITKH
                     VAQILDSRMNTKYDENDKLIREVKVITLKSKLVSDFRKDFQFYKVREINNYHHAHDAYL
                     NAVVGTALIKKYPKLESEFVYGDYKVYDVRKMIAKSEQEIGKATAKYFFYSNIMNFFKT
                     EITLANGEIRKRPLIETNGETGEIVWDKGRDFATVRKVLSMPQVNIVKKTEVQTGGFSK
                     ESILPKRNSDKLIARKKDWDPKKYGGFDSPTVAYSVLVVAKVEKGKSKKLKSVKELLGI
                     TIMERSSFEKNPIDFLEAKGYKEVKKDLIIKLPKYSLFELENGRKRMLASAGELQKGNE
                     LALPSKYVNFLYLASHYEKLKGSPEDNEQKQLFVEQHKHYLDEIIEQISEFSKRVILAD
                     ANLDKVLSAYNKHRDKPIREQAENIIHLFTLTNLGAPAAFKYFDTTIDRKRYTSTKEVL
                     DATLIHQSITGLYETRIDLSQLGGD"
     CDS             6991..7038
                     /codon_start=1
                     /product="bipartite nuclear localization signal from 
                     nucleoplasmin"
                     /label=nucleoplasmin NLS
                     /translation="KRPAATKKAGQAKKKK"
     CDS             7039..7062
                     /codon_start=1
                     /product="FLAG(R) epitope tag, followed by an enterokinase 
                     cleavage site"
                     /label=FLAG
                     /translation="DYKDDDDK"
     CDS             7072..7128
                     /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             7129..7725
                     /codon_start=1
                     /gene="pac from Streptomyces alboniger"
                     /product="puromycin N-acetyltransferase"
                     /label=PuroR
                     /note="confers resistance to puromycin"
                     /translation="TEYKPTVRLATRDDVPRAVRTLAAAFADYPATRHTVDPDRHIERV
                     TELQELFLTRVGLDIGKVWVADDGAAVAVWTTPESVEAGAVFAEIGPRMAELSGSRLAA
                     QQQMEGLLAPHRPKEPAWFLATVGVSPDHQGKGLGSAVVLPGVEAAERAGVPAFLETSA
                     PRNLPFYERLGFTVTADVEVPEGPRTWCMTRKPGA"
     primer_bind     7622..7642
                     /label=Puro-F
                     /note="Puromycin resistance gene, forward primer"
     misc_feature    7741..8329
                     /label=WPRE
                     /note="woodchuck hepatitis virus posttranscriptional 
                     regulatory element"
     primer_bind     complement(7794..7814)
                     /label=WPRE-R
                     /note="WPRE, reverse primer"
     CDS             complement(8212..8223)
                     /codon_start=1
                     /product="Factor Xa recognition and cleavage site"
                     /label=Factor Xa site
                     /translation="IEGR"
     LTR             8401..8634
                     /label=3' LTR (Delta-U3)
                     /note="self-inactivating 3' long terminal repeat (LTR) from
                     HIV-1"
     primer_bind     complement(8660..8677)
                     /label=BGH-rev
                     /note="Bovine growth hormone terminator, reverse primer. 
                     Also called BGH reverse"
     polyA_signal    8666..8890
                     /label=bGH poly(A) signal
                     /note="bovine growth hormone polyadenylation signal"
     rep_origin      8936..9364
                     /direction=RIGHT
                     /label=f1 ori
                     /note="f1 bacteriophage origin of replication; arrow 
                     indicates direction of (+) strand synthesis"
     primer_bind     complement(9023..9042)
                     /label=F1ori-R
                     /note="F1 origin, reverse primer"
     primer_bind     9233..9254
                     /label=F1ori-F
                     /note="F1 origin, forward primer"
     primer_bind     complement(9373..9393)
                     /label=pBABE 3'
                     /note="SV40 enhancer, reverse primer for pBABE vectors"
     promoter        9378..9707
                     /label=SV40 promoter
                     /note="SV40 enhancer and early promoter"
     rep_origin      9558..9693
                     /label=SV40 ori
                     /note="SV40 origin of replication"
     primer_bind     9620..9639
                     /label=SV40pro-F
                     /note="SV40 promoter/origin, forward primer"
     promoter        9755..9802
                     /label=EM7 promoter
                     /note="synthetic bacterial promoter "
     CDS             9821..10195
                     /codon_start=1
                     /gene="Sh ble from Streptoalloteichus hindustanus"
                     /product="antibiotic-binding protein"
                     /label=BleoR
                     /note="confers resistance to bleomycin, phleomycin, and 
                     Zeocin(TM)"
                     /translation="MAKLTSAVPVLTARDVAGAVEFWTDRLGFSRDFVEDDFAGVVRDD
                     VTLFISAVQDQVVPDNTLAWVWVRGLDELYAEWSEVVSTNFRDASGPAMTEIGEQPWGR
                     EFALRDPAGNCVHFVAEEQD"
     polyA_signal    10325..10446
                     /label=SV40 poly(A) signal
                     /note="SV40 polyadenylation signal"
     primer_bind     complement(10362..10381)
                     /label=SV40pA-R
                     /note="SV40 polyA, reverse primer"
     primer_bind     10416..10435
                     /label=EBV-rev
                     /note="SV40 polyA terminator, reverse primer"
     primer_bind     complement(10495..10511)
                     /label=M13 rev
                     /note="common sequencing primer, one of multiple similar 
                     variants"
     primer_bind     complement(10495..10511)
                     /label=M13 Reverse
                     /note="In lacZ gene. Also called M13-rev"
     primer_bind     complement(10508..10530)
                     /label=M13/pUC Reverse
                     /note="In lacZ gene"
     protein_bind    10519..10535
                     /label=lac operator
                     /bound_moiety="lac repressor encoded by lacI"
                     /note="The lac repressor binds to the lac operator to 
                     inhibit transcription in E. coli. This inhibition can be 
                     relieved by adding lactose or 
                     isopropyl-beta-D-thiogalactopyranoside (IPTG)."
     promoter        complement(join(10543..10549,10550..10567,10568..10573))
                     /label=lac promoter
                     /note="promoter for the E. coli lac operon"
     protein_bind    10588..10609
                     /label=CAP binding site
                     /bound_moiety="E. coli catabolite activator protein"
                     /note="CAP binding activates transcription in the presence 
                     of cAMP."
     primer_bind     complement(10726..10743)
                     /label=L4440
                     /note="L4440 vector, forward primer"
     rep_origin      complement(10897..11485)
                     /direction=LEFT
                     /label=ori
                     /note="high-copy-number ColE1/pMB1/pBR322/pUC origin of 
                     replication"
     primer_bind     complement(10977..10996)
                     /label=pBR322ori-F
                     /note="pBR322 origin, forward primer"
     CDS             complement(join(11656..12447,12448..12516))
                     /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     12279..12298
                     /label=Amp-R
                     /note="Ampicillin resistance gene, reverse primer"
     promoter        complement(12517..12621)
                     /gene="bla"
                     /label=AmpR promoter
ORIGIN
        1 gtcgacggat cgggagatct cccgatcccc tatggtgcac tctcagtaca atctgctctg
       61 atgccgcata gttaagccag tatctgctcc ctgcttgtgt gttggaggtc gctgagtagt
      121 gcgcgagcaa aatttaagct acaacaaggc aaggcttgac cgacaattgc atgaagaatc
      181 tgcttagggt taggcgtttt gcgctgcttc gcgatgtacg ggccagatat acgcgttgac
      241 attgattatt gactagttat taatagtaat caattacggg gtcattagtt catagcccat
      301 atatggagtt ccgcgttaca taacttacgg taaatggccc gcctggctga ccgcccaacg
      361 acccccgccc attgacgtca ataatgacgt atgttcccat agtaacgcca atagggactt
      421 tccattgacg tcaatgggtg gagtatttac ggtaaactgc ccacttggca gtacatcaag
      481 tgtatcatat gccaagtacg ccccctattg acgtcaatga cggtaaatgg cccgcctggc
      541 attatgccca gtacatgacc ttatgggact ttcctacttg gcagtacatc tacgtattag
      601 tcatcgctat taccatggtg atgcggtttt ggcagtacat caatgggcgt ggatagcggt
      661 ttgactcacg gggatttcca agtctccacc ccattgacgt caatgggagt ttgttttggc
      721 accaaaatca acgggacttt ccaaaatgtc gtaacaactc cgccccattg acgcaaatgg
      781 gcggtaggcg tgtacggtgg gaggtctata taagcagcgc gttttgcctg tactgggtct
      841 ctctggttag accagatctg agcctgggag ctctctggct aactagggaa cccactgctt
      901 aagcctcaat aaagcttgcc ttgagtgctt caagtagtgt gtgcccgtct gttgtgtgac
      961 tctggtaact agagatccct cagacccttt tagtcagtgt ggaaaatctc tagcagtggc
     1021 gcccgaacag ggacttgaaa gcgaaaggga aaccagagga gctctctcga cgcaggactc
     1081 ggcttgctga agcgcgcacg gcaagaggcg aggggcggcg actggtgagt acgccaaaaa
     1141 ttttgactag cggaggctag aaggagagag atgggtgcga gagcgtcagt attaagcggg
     1201 ggagaattag atcgcgatgg gaaaaaattc ggttaaggcc agggggaaag aaaaaatata
     1261 aattaaaaca tatagtatgg gcaagcaggg agctagaacg attcgcagtt aatcctggcc
     1321 tgttagaaac atcagaaggc tgtagacaaa tactgggaca gctacaacca tcccttcaga
     1381 caggatcaga agaacttaga tcattatata atacagtagc aaccctctat tgtgtgcatc
     1441 aaaggataga gataaaagac accaaggaag ctttagacaa gatagaggaa gagcaaaaca
     1501 aaagtaagac caccgcacag caagcggccg ctgatcttca gacctggagg aggagatatg
     1561 agggacaatt ggagaagtga attatataaa tataaagtag taaaaattga accattagga
     1621 gtagcaccca ccaaggcaaa gagaagagtg gtgcagagag aaaaaagagc agtgggaata
     1681 ggagctttgt tccttgggtt cttgggagca gcaggaagca ctatgggcgc agcgtcaatg
     1741 acgctgacgg tacaggccag acaattattg tctggtatag tgcagcagca gaacaatttg
     1801 ctgagggcta ttgaggcgca acagcatctg ttgcaactca cagtctgggg catcaagcag
     1861 ctccaggcaa gaatcctggc tgtggaaaga tacctaaagg atcaacagct cctggggatt
     1921 tggggttgct ctggaaaact catttgcacc actgctgtgc cttggaatgc tagttggagt
     1981 aataaatctc tggaacagat ttggaatcac acgacctgga tggagtggga cagagaaatt
     2041 aacaattaca caagcttaat acactcctta attgaagaat cgcaaaacca gcaagaaaag
     2101 aatgaacaag aattattgga attagataaa tgggcaagtt tgtggaattg gtttaacata
     2161 acaaattggc tgtggtatat aaaattattc ataatgatag taggaggctt ggtaggttta
     2221 agaatagttt ttgctgtact ttctatagtg aatagagtta ggcagggata ttcaccatta
     2281 tcgtttcaga cccacctccc aaccccgagg ggacccgaca ggcccgaagg aatagaagaa
     2341 gaaggtggag agagagacag agacagatcc attcgattag tgaacggatc ggcactgcgt
     2401 gcgccaattc tgcagacaaa tggcagtatt catccacaat tttaaaagaa aaggggggat
     2461 tggggggtac agtgcagggg aaagaatagt agacataata gcaacagaca tacaaactaa
     2521 agaattacaa aaacaaatta caaaaattca aaattttcgg gtttattaca gggacagcag
     2581 agatccagtt tggttaatta agctagctag gtcttgaaag gagtgggaat tggctccggt
     2641 gcccgtcagt gggcagagcg cacatcgccc acagtccccg agaagttggg gggaggggtc
     2701 ggcaattgat ccggtgccta gagaaggtgg cgcggggtaa actgggaaag tgatgtcgtg
     2761 tactggctcc gcctttttcc cgagggtggg ggagaaccgt atataagtgc agtagtcgcc
     2821 gtgaacgttc tttttcgcaa cgggtttgcc gccagaacac aggaccggtt ctagagcgct
     2881 gccaccatgg acaagaagta cagcatcggc ctggacatcg gcaccaactc tgtgggctgg
     2941 gccgtgatca ccgacgagta caaggtgccc agcaagaaat tcaaggtgct gggcaacacc
     3001 gaccggcaca gcatcaagaa gaacctgatc ggagccctgc tgttcgacag cggcgaaaca
     3061 gccgaggcca cccggctgaa gagaaccgcc agaagaagat acaccagacg gaagaaccgg
     3121 atctgctatc tgcaagagat cttcagcaac gagatggcca aggtggacga cagcttcttc
     3181 cacagactgg aagagtcctt cctggtggaa gaggataaga agcacgagcg gcaccccatc
     3241 ttcggcaaca tcgtggacga ggtggcctac cacgagaagt accccaccat ctaccacctg
     3301 agaaagaaac tggtggacag caccgacaag gccgacctgc ggctgatcta tctggccctg
     3361 gcccacatga tcaagttccg gggccacttc ctgatcgagg gcgacctgaa ccccgacaac
     3421 agcgacgtgg acaagctgtt catccagctg gtgcagacct acaaccagct gttcgaggaa
     3481 aaccccatca acgccagcgg cgtggacgcc aaggccatcc tgtctgccag actgagcaag
     3541 agcagacggc tggaaaatct gatcgcccag ctgcccggcg agaagaagaa tggcctgttc
     3601 ggaaacctga ttgccctgag cctgggcctg acccccaact tcaagagcaa cttcgacctg
     3661 gccgaggatg ccaaactgca gctgagcaag gacacctacg acgacgacct ggacaacctg
     3721 ctggcccaga tcggcgacca gtacgccgac ctgtttctgg ccgccaagaa cctgtccgac
     3781 gccatcctgc tgagcgacat cctgagagtg aacaccgaga tcaccaaggc ccccctgagc
     3841 gcctctatga tcaagagata cgacgagcac caccaggacc tgaccctgct gaaagctctc
     3901 gtgcggcagc agctgcctga gaagtacaaa gagattttct tcgaccagag caagaacggc
     3961 tacgccggct acattgacgg cggagccagc caggaagagt tctacaagtt catcaagccc
     4021 atcctggaaa agatggacgg caccgaggaa ctgctcgtga agctgaacag agaggacctg
     4081 ctgcggaagc agcggacctt cgacaacggc agcatccccc accagatcca cctgggagag
     4141 ctgcacgcca ttctgcggcg gcaggaagat ttttacccat tcctgaagga caaccgggaa
     4201 aagatcgaga agatcctgac cttccgcatc ccctactacg tgggccctct ggccagggga
     4261 aacagcagat tcgcctggat gaccagaaag agcgaggaaa ccatcacccc ctggaacttc
     4321 gaggaagtgg tggacaaggg cgcttccgcc cagagcttca tcgagcggat gaccaacttc
     4381 gataagaacc tgcccaacga gaaggtgctg cccaagcaca gcctgctgta cgagtacttc
     4441 accgtgtata acgagctgac caaagtgaaa tacgtgaccg agggaatgag aaagcccgcc
     4501 ttcctgagcg gcgagcagaa aaaggccatc gtggacctgc tgttcaagac caaccggaaa
     4561 gtgaccgtga agcagctgaa agaggactac ttcaagaaaa tcgagtgctt cgactccgtg
     4621 gaaatctccg gcgtggaaga tcggttcaac gcctccctgg gcacatacca cgatctgctg
     4681 aaaattatca aggacaagga cttcctggac aatgaggaaa acgaggacat tctggaagat
     4741 atcgtgctga ccctgacact gtttgaggac agagagatga tcgaggaacg gctgaaaacc
     4801 tatgcccacc tgttcgacga caaagtgatg aagcagctga agcggcggag atacaccggc
     4861 tggggcaggc tgagccggaa gctgatcaac ggcatccggg acaagcagtc cggcaagaca
     4921 atcctggatt tcctgaagtc cgacggcttc gccaacagaa acttcatgca gctgatccac
     4981 gacgacagcc tgacctttaa agaggacatc cagaaagccc aggtgtccgg ccagggcgat
     5041 agcctgcacg agcacattgc caatctggcc ggcagccccg ccattaagaa gggcatcctg
     5101 cagacagtga aggtggtgga cgagctcgtg aaagtgatgg gccggcacaa gcccgagaac
     5161 atcgtgatcg aaatggccag agagaaccag accacccaga agggacagaa gaacagccgc
     5221 gagagaatga agcggatcga agagggcatc aaagagctgg gcagccagat cctgaaagaa
     5281 caccccgtgg aaaacaccca gctgcagaac gagaagctgt acctgtacta cctgcagaat
     5341 gggcgggata tgtacgtgga ccaggaactg gacatcaacc ggctgtccga ctacgatgtg
     5401 gaccatatcg tgcctcagag ctttctgaag gacgactcca tcgacaacaa ggtgctgacc
     5461 agaagcgaca agaaccgggg caagagcgac aacgtgccct ccgaagaggt cgtgaagaag
     5521 atgaagaact actggcggca gctgctgaac gccaagctga ttacccagag aaagttcgac
     5581 aatctgacca aggccgagag aggcggcctg agcgaactgg ataaggccgg cttcatcaag
     5641 agacagctgg tggaaacccg gcagatcaca aagcacgtgg cacagatcct ggactcccgg
     5701 atgaacacta agtacgacga gaatgacaag ctgatccggg aagtgaaagt gatcaccctg
     5761 aagtccaagc tggtgtccga tttccggaag gatttccagt tttacaaagt gcgcgagatc
     5821 aacaactacc accacgccca cgacgcctac ctgaacgccg tcgtgggaac cgccctgatc
     5881 aaaaagtacc ctaagctgga aagcgagttc gtgtacggcg actacaaggt gtacgacgtg
     5941 cggaagatga tcgccaagag cgagcaggaa atcggcaagg ctaccgccaa gtacttcttc
     6001 tacagcaaca tcatgaactt tttcaagacc gagattaccc tggccaacgg cgagatccgg
     6061 aagcggcctc tgatcgagac aaacggcgaa accggggaga tcgtgtggga taagggccgg
     6121 gattttgcca ccgtgcggaa agtgctgagc atgccccaag tgaatatcgt gaaaaagacc
     6181 gaggtgcaga caggcggctt cagcaaagag tctatcctgc ccaagaggaa cagcgataag
     6241 ctgatcgcca gaaagaagga ctgggaccct aagaagtacg gcggcttcga cagccccacc
     6301 gtggcctatt ctgtgctggt ggtggccaaa gtggaaaagg gcaagtccaa gaaactgaag
     6361 agtgtgaaag agctgctggg gatcaccatc atggaaagaa gcagcttcga gaagaatccc
     6421 atcgactttc tggaagccaa gggctacaaa gaagtgaaaa aggacctgat catcaagctg
     6481 cctaagtact ccctgttcga gctggaaaac ggccggaaga gaatgctggc ctctgccggc
     6541 gaactgcaga agggaaacga actggccctg ccctccaaat atgtgaactt cctgtacctg
     6601 gccagccact atgagaagct gaagggctcc cccgaggata atgagcagaa acagctgttt
     6661 gtggaacagc acaagcacta cctggacgag atcatcgagc agatcagcga gttctccaag
     6721 agagtgatcc tggccgacgc taatctggac aaagtgctgt ccgcctacaa caagcaccgg
     6781 gataagccca tcagagagca ggccgagaat atcatccacc tgtttaccct gaccaatctg
     6841 ggagcccctg ccgccttcaa gtactttgac accaccatcg accggaagag gtacaccagc
     6901 accaaagagg tgctggacgc caccctgatc caccagagca tcaccggcct gtacgagaca
     6961 cggatcgacc tgtctcagct gggaggcgac aagcgacctg ccgccacaaa gaaggctgga
     7021 caggctaaga agaagaaaga ttacaaagac gatgacgata agggatccgg cgcaacaaac
     7081 ttctctctgc tgaaacaagc cggagatgtc gaagagaatc ctggaccgac cgagtacaag
     7141 cccacggtgc gcctcgccac ccgcgacgac gtccccaggg ccgtacgcac cctcgccgcc
     7201 gcgttcgccg actaccccgc cacgcgccac accgtcgatc cggaccgcca catcgagcgg
     7261 gtcaccgagc tgcaagaact cttcctcacg cgcgtcgggc tcgacatcgg caaggtgtgg
     7321 gtcgcggacg acggcgccgc ggtggcggtc tggaccacgc cggagagcgt cgaagcgggg
     7381 gcggtgttcg ccgagatcgg cccgcgcatg gccgagttga gcggttcccg gctggccgcg
     7441 cagcaacaga tggaaggcct cctggcgccg caccggccca aggagcccgc gtggttcctg
     7501 gccaccgtcg gagtctcgcc cgaccaccag ggcaagggtc tgggcagcgc cgtcgtgctc
     7561 cccggagtgg aggcggccga gcgcgccggg gtgcccgcct tcctggagac ctccgcgccc
     7621 cgcaacctcc ccttctacga gcggctcggc ttcaccgtca ccgccgacgt cgaggtgccc
     7681 gaaggaccgc gcacctggtg catgacccgc aagcccggtg cctgaacgcg ttaagtcgac
     7741 aatcaacctc tggattacaa aatttgtgaa agattgactg gtattcttaa ctatgttgct
     7801 ccttttacgc tatgtggata cgctgcttta atgcctttgt atcatgctat tgcttcccgt
     7861 atggctttca ttttctcctc cttgtataaa tcctggttgc tgtctcttta tgaggagttg
     7921 tggcccgttg tcaggcaacg tggcgtggtg tgcactgtgt ttgctgacgc aacccccact
     7981 ggttggggca ttgccaccac ctgtcagctc ctttccggga ctttcgcttt ccccctccct
     8041 attgccacgg cggaactcat cgccgcctgc cttgcccgct gctggacagg ggctcggctg
     8101 ttgggcactg acaattccgt ggtgttgtcg gggaaatcat cgtcctttcc ttggctgctc
     8161 gcctgtgttg ccacctggat tctgcgcggg acgtccttct gctacgtccc ttcggccctc
     8221 aatccagcgg accttccttc ccgcggcctg ctgccggctc tgcggcctct tccgcgtctt
     8281 cgccttcgcc ctcagacgag tcggatctcc ctttgggccg cctccccgcg tcgactttaa
     8341 gaccaatgac ttacaaggca gctgtagatc ttagccactt tttaaaagaa aaggggggac
     8401 tggaagggct aattcactcc caacgaagac aagatctgct ttttgcttgt actgggtctc
     8461 tctggttaga ccagatctga gcctgggagc tctctggcta actagggaac ccactgctta
     8521 agcctcaata aagcttgcct tgagtgcttc aagtagtgtg tgcccgtctg ttgtgtgact
     8581 ctggtaacta gagatccctc agaccctttt agtcagtgtg gaaaatctct agcagggccc
     8641 gtttaaaccc gctgatcagc ctcgactgtg ccttctagtt gccagccatc tgttgtttgc
     8701 ccctcccccg tgccttcctt gaccctggaa ggtgccactc ccactgtcct ttcctaataa
     8761 aatgaggaaa ttgcatcgca ttgtctgagt aggtgtcatt ctattctggg gggtggggtg
     8821 gggcaggaca gcaaggggga ggattgggaa gacaatagca ggcatgctgg ggatgcggtg
     8881 ggctctatgg cttctgaggc ggaaagaacc agctggggct ctagggggta tccccacgcg
     8941 ccctgtagcg gcgcattaag cgcggcgggt gtggtggtta cgcgcagcgt gaccgctaca
     9001 cttgccagcg ccctagcgcc cgctcctttc gctttcttcc cttcctttct cgccacgttc
     9061 gccggctttc cccgtcaagc tctaaatcgg gggctccctt tagggttccg atttagtgct
     9121 ttacggcacc tcgaccccaa aaaacttgat tagggtgatg gttcacgtag tgggccatcg
     9181 ccctgataga cggtttttcg ccctttgacg ttggagtcca cgttctttaa tagtggactc
     9241 ttgttccaaa ctggaacaac actcaaccct atctcggtct attcttttga tttataaggg
     9301 attttgccga tttcggccta ttggttaaaa aatgagctga tttaacaaaa atttaacgcg
     9361 aattaattct gtggaatgtg tgtcagttag ggtgtggaaa gtccccaggc tccccagcag
     9421 gcagaagtat gcaaagcatg catctcaatt agtcagcaac caggtgtgga aagtccccag
     9481 gctccccagc aggcagaagt atgcaaagca tgcatctcaa ttagtcagca accatagtcc
     9541 cgcccctaac tccgcccatc ccgcccctaa ctccgcccag ttccgcccat tctccgcccc
     9601 atggctgact aatttttttt atttatgcag aggccgaggc cgcctctgcc tctgagctat
     9661 tccagaagta gtgaggaggc ttttttggag gcctaggctt ttgcaaaaag ctcccgggag
     9721 cttgtatatc cattttcgga tctgatcagc acgtgttgac aattaatcat cggcatagta
     9781 tatcggcata gtataatacg acaaggtgag gaactaaacc atggccaagt tgaccagtgc
     9841 cgttccggtg ctcaccgcgc gcgacgtcgc cggagcggtc gagttctgga ccgaccggct
     9901 cgggttctcc cgggacttcg tggaggacga cttcgccggt gtggtccggg acgacgtgac
     9961 cctgttcatc agcgcggtcc aggaccaggt ggtgccggac aacaccctgg cctgggtgtg
    10021 ggtgcgcggc ctggacgagc tgtacgccga gtggtcggag gtcgtgtcca cgaacttccg
    10081 ggacgcctcc gggccggcca tgaccgagat cggcgagcag ccgtgggggc gggagttcgc
    10141 cctgcgcgac ccggccggca actgcgtgca cttcgtggcc gaggagcagg actgacacgt
    10201 gctacgagat ttcgattcca ccgccgcctt ctatgaaagg ttgggcttcg gaatcgtttt
    10261 ccgggacgcc ggctggatga tcctccagcg cggggatctc atgctggagt tcttcgccca
    10321 ccccaacttg tttattgcag cttataatgg ttacaaataa agcaatagca tcacaaattt
    10381 cacaaataaa gcattttttt cactgcattc tagttgtggt ttgtccaaac tcatcaatgt
    10441 atcttatcat gtctgtatac cgtcgacctc tagctagagc ttggcgtaat catggtcata
    10501 gctgtttcct gtgtgaaatt gttatccgct cacaattcca cacaacatac gagccggaag
    10561 cataaagtgt aaagcctggg gtgcctaatg agtgagctaa ctcacattaa ttgcgttgcg
    10621 ctcactgccc gctttccagt cgggaaacct gtcgtgccag ctgcattaat gaatcggcca
    10681 acgcgcgggg agaggcggtt tgcgtattgg gcgctcttcc gcttcctcgc tcactgactc
    10741 gctgcgctcg gtcgttcggc tgcggcgagc ggtatcagct cactcaaagg cggtaatacg
    10801 gttatccaca gaatcagggg ataacgcagg aaagaacatg tgagcaaaag gccagcaaaa
    10861 ggccaggaac cgtaaaaagg ccgcgttgct ggcgtttttc cataggctcc gcccccctga
    10921 cgagcatcac aaaaatcgac gctcaagtca gaggtggcga aacccgacag gactataaag
    10981 ataccaggcg tttccccctg gaagctccct cgtgcgctct cctgttccga ccctgccgct
    11041 taccggatac ctgtccgcct ttctcccttc gggaagcgtg gcgctttctc atagctcacg
    11101 ctgtaggtat ctcagttcgg tgtaggtcgt tcgctccaag ctgggctgtg tgcacgaacc
    11161 ccccgttcag cccgaccgct gcgccttatc cggtaactat cgtcttgagt ccaacccggt
    11221 aagacacgac ttatcgccac tggcagcagc cactggtaac aggattagca gagcgaggta
    11281 tgtaggcggt gctacagagt tcttgaagtg gtggcctaac tacggctaca ctagaagaac
    11341 agtatttggt atctgcgctc tgctgaagcc agttaccttc ggaaaaagag ttggtagctc
    11401 ttgatccggc aaacaaacca ccgctggtag cggtggtttt tttgtttgca agcagcagat
    11461 tacgcgcaga aaaaaaggat ctcaagaaga tcctttgatc ttttctacgg ggtctgacgc
    11521 tcagtggaac gaaaactcac gttaagggat tttggtcatg agattatcaa aaaggatctt
    11581 cacctagatc cttttaaatt aaaaatgaag ttttaaatca atctaaagta tatatgagta
    11641 aacttggtct gacagttacc aatgcttaat cagtgaggca cctatctcag cgatctgtct
    11701 atttcgttca tccatagttg cctgactccc cgtcgtgtag ataactacga tacgggaggg
    11761 cttaccatct ggccccagtg ctgcaatgat accgcgagac ccacgctcac cggctccaga
    11821 tttatcagca ataaaccagc cagccggaag ggccgagcgc agaagtggtc ctgcaacttt
    11881 atccgcctcc atccagtcta ttaattgttg ccgggaagct agagtaagta gttcgccagt
    11941 taatagtttg cgcaacgttg ttgccattgc tacaggcatc gtggtgtcac gctcgtcgtt
    12001 tggtatggct tcattcagct ccggttccca acgatcaagg cgagttacat gatcccccat
    12061 gttgtgcaaa aaagcggtta gctccttcgg tcctccgatc gttgtcagaa gtaagttggc
    12121 cgcagtgtta tcactcatgg ttatggcagc actgcataat tctcttactg tcatgccatc
    12181 cgtaagatgc ttttctgtga ctggtgagta ctcaaccaag tcattctgag aatagtgtat
    12241 gcggcgaccg agttgctctt gcccggcgtc aatacgggat aataccgcgc cacatagcag
    12301 aactttaaaa gtgctcatca ttggaaaacg ttcttcgggg cgaaaactct caaggatctt
    12361 accgctgttg agatccagtt cgatgtaacc cactcgtgca cccaactgat cttcagcatc
    12421 ttttactttc accagcgttt ctgggtgagc aaaaacagga aggcaaaatg ccgcaaaaaa
    12481 gggaataagg gcgacacgga aatgttgaat actcatactc ttcctttttc aatattattg
    12541 aagcatttat cagggttatt gtctcatgag cggatacata tttgaatgta tttagaaaaa
    12601 taaacaaata ggggttccgc gcacatttcc ccgaaaagtg ccacctgac
//
