LOCUS       sequence-454333-       13548 bp    DNA     circular SYN 15-DEC-2025
DEFINITION  mU6-driven expression of spCas9 sgRNAs with 
            EF1a-SpCas9-2A-Puro-2A-mCherry.
ACCESSION   .
VERSION     .
KEYWORDS    .
SOURCE      synthetic DNA construct
  ORGANISM  synthetic DNA construct
REFERENCE   1  (bases 1 to 13548)
  AUTHORS   El Kassem G, Hillmer J, Boettcher M
  TITLE     Evaluation of Cas13d as a tool for genetic interaction mapping.
  JOURNAL   Nat Commun. 2025 Feb 14;16(1):1631. doi: 10.1038/s41467-025-56747-4.
  PUBMED    39952934
REFERENCE   2  (bases 1 to 13548)
  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: "Nat 
            Commun."; date: "2025-02-14"; volume: "16(1):1631. doi"; pages: " 
            10.1038/s41467-025-56747-4"
COMMENT     Sequence Label: pMB1: All in one SpCas9 expression and sgRNA c...
FEATURES             Location/Qualifiers
     source          1..13548
                     /mol_type="other DNA"
                     /organism="synthetic DNA construct"
     CDS             1023..1070
                     /codon_start=1
                     /product="bipartite nuclear localization signal from 
                     nucleoplasmin"
                     /label=nucleoplasmin NLS
                     /translation="KRPAATKKAGQAKKKK"
     CDS             1071..1094
                     /codon_start=1
                     /product="FLAG(R) epitope tag, followed by an enterokinase 
                     cleavage site"
                     /label=FLAG
                     /translation="DYKDDDDK"
     CDS             1104..1160
                     /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             1161..1754
                     /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     1654..1674
                     /label=Puro-F
                     /note="Puromycin resistance gene, forward primer"
     CDS             1764..1817
                     /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"
     regulatory      1824..1833
                     /label=Kozak sequence
                     /note="vertebrate consensus sequence for strong initiation 
                     of translation (Kozak, 1987)"
                     /regulatory_class="other"
     CDS             1830..2537
                     /codon_start=1
                     /product="monomeric derivative of DsRed fluorescent protein
                     (Shaner et al., 2004)"
                     /label=mCherry
                     /note="mammalian codon-optimized"
                     /translation="MVSKGEEDNMAIIKEFMRFKVHMEGSVNGHEFEIEGEGEGRPYEG
                     TQTAKLKVTKGGPLPFAWDILSPQFMYGSKAYVKHPADIPDYLKLSFPEGFKWERVMNF
                     EDGGVVTVTQDSSLQDGEFIYKVKLRGTNFPSDGPVMQKKTMGWEASSERMYPEDGALK
                     GEIKQRLKLKDGGHYDAEVKTTYKAKKPVQLPGAYNVNIKLDITSHNEDYTIVEQYERA
                     EGRHSTGGMDELYK"
     primer_bind     complement(1975..1993)
                     /label=mCherry-R
                     /note="mCherry, reverse primer"
     primer_bind     2243..2262
                     /label=mCherry-F
                     /note="mCherry, forward primer"
     CDS             2547..2567
                     /codon_start=1
                     /product="nuclear localization signal of SV40 (simian virus
                     40) large T antigen"
                     /label=SV40 NLS
                     /translation="PKKKRKV"
     misc_feature    2647..3235
                     /label=WPRE
                     /note="woodchuck hepatitis virus posttranscriptional 
                     regulatory element"
     primer_bind     complement(2700..2720)
                     /label=WPRE-R
                     /note="WPRE, reverse primer"
     CDS             complement(3118..3129)
                     /codon_start=1
                     /product="Factor Xa recognition and cleavage site"
                     /label=Factor Xa site
                     /translation="IEGR"
     primer_bind     complement(3238..3254)
                     /label=KS primer
                     /note="common sequencing primer, one of multiple similar 
                     variants"
     primer_bind     complement(3239..3255)
                     /label=pBluescriptKS
                     /note="For pBluescript vector"
     LTR             3764..3944
                     /label=5' LTR (truncated)
                     /note="truncated 5' long terminal repeat (LTR) from HIV-1"
     rep_origin      complement(4006..4594)
                     /direction=LEFT
                     /label=ori
                     /note="high-copy-number ColE1/pMB1/pBR322/pUC origin of 
                     replication"
     primer_bind     complement(4086..4105)
                     /label=pBR322ori-F
                     /note="pBR322 origin, forward primer"
     CDS             complement(join(4765..5556,5557..5625))
                     /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     5388..5407
                     /label=Amp-R
                     /note="Ampicillin resistance gene, reverse primer"
     promoter        complement(5626..5730)
                     /gene="bla"
                     /label=AmpR promoter
     primer_bind     complement(5805..5824)
                     /label=pRS-marker
                     /note="pRS vectors, use to sequence yeast selectable 
                     marker"
     enhancer        5996..6375
                     /label=CMV enhancer
                     /note="human cytomegalovirus immediate early enhancer"
     promoter        6377..6575
                     /label=CMV promoter
                     /note="human cytomegalovirus (CMV) immediate early 
                     promoter"
     primer_bind     6530..6550
                     /label=CMV-F
                     /note="Human CMV immediate early promoter, forward primer"
     LTR             6593..6773
                     /label=5' LTR (truncated)
                     /note="truncated 5' long terminal repeat (LTR) from HIV-1"
     misc_feature    6820..6945
                     /label=HIV-1 Psi
                     /note="packaging signal of human immunodeficiency virus 
                     type 1"
     misc_feature    7442..7675
                     /label=RRE
                     /note="The Rev response element (RRE) of HIV-1 allows for 
                     Rev-dependent mRNA export from the nucleus to the 
                     cytoplasm."
     CDS             7860..7904
                     /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    8202..8319
                     /label=cPPT/CTS
                     /note="central polypurine tract and central termination 
                     sequence of HIV-1"
     protein_bind    complement(8572..8605)
                     /label=loxP
                     /bound_moiety="Cre recombinase"
                     /note="Cre-mediated recombination occurs in the 8-bp core 
                     sequence (ATGTATGC) (Shaw et al., 2021)."
     primer_bind     8609..8629
                     /label=mU6-F
                     /note="Mouse U6 promoter, forward primer"
     promoter        10231..10442
                     /label=EF-1-alpha core promoter
                     /note="core promoter for human elongation factor 
                     EF-1-alpha"
     regulatory      10461..10470
                     /label=Kozak sequence
                     /note="vertebrate consensus sequence for strong initiation 
                     of translation (Kozak, 1987)"
                     /regulatory_class="other"
     CDS             join(10467..13548,1..1022)
                     /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             join(10467..13548,1..1022)
                     /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"
ORIGIN
        1 aaatcggcaa ggctaccgcc aagtacttct tctacagcaa catcatgaac tttttcaaga
       61 ccgagattac cctggccaac ggcgagatcc ggaagcggcc tctgatcgag acaaacggcg
      121 aaaccgggga gatcgtgtgg gataagggcc gggattttgc caccgtgcgg aaagtgctga
      181 gcatgcccca agtgaatatc gtgaaaaaga ccgaggtgca gacaggcggc ttcagcaaag
      241 agtctatcct gcccaagagg aacagcgata agctgatcgc cagaaagaag gactgggacc
      301 ctaagaagta cggcggcttc gacagcccca ccgtggccta ttctgtgctg gtggtggcca
      361 aagtggaaaa gggcaagtcc aagaaactga agagtgtgaa agagctgctg gggatcacca
      421 tcatggaaag aagcagcttc gagaagaatc ccatcgactt tctggaagcc aagggctaca
      481 aagaagtgaa aaaggacctg atcatcaagc tgcctaagta ctccctgttc gagctggaaa
      541 acggccggaa gagaatgctg gcctctgccg gcgaactgca gaagggaaac gaactggccc
      601 tgccctccaa atatgtgaac ttcctgtacc tggccagcca ctatgagaag ctgaagggct
      661 cccccgagga taatgagcag aaacagctgt ttgtggaaca gcacaagcac tacctggatg
      721 agatcatcga gcagatcagc gagttctcca agagagtgat cctggccgac gctaatctgg
      781 acaaagtgct gtccgcctac aacaagcacc gggataagcc catcagagag caggccgaga
      841 atatcatcca cctgtttacc ctgaccaatc tgggagcccc tgccgccttc aagtactttg
      901 acaccaccat cgaccggaag aggtacacca gcaccaaaga ggtgctggac gccaccctga
      961 tccaccagag catcaccggc ctgtacgaga cacggatcga cctgtctcag ctgggaggcg
     1021 acaagcgacc tgccgccaca aagaaggctg gacaggctaa gaagaagaaa gattacaaag
     1081 acgatgacga taagggatcc ggcgcaacaa acttctctct gctgaaacaa gccggagatg
     1141 tcgaagagaa tcctggaccg accgagtaca agcccacggt gcgcctcgcc acccgcgacg
     1201 acgtccccag ggccgtacgc accctcgccg ccgcgttcgc cgactacccc gccacgcgcc
     1261 acaccgtcga cccggaccgc cacatcgagc gggtcaccga gctgcaagaa ctcttcctca
     1321 cgcgcgtcgg gctcgacatc ggcaaggtgt gggtcgcgga cgacggcgcc gcggtggcgg
     1381 tctggaccac gccggagagc gtcgaagcgg gggcggtgtt cgccgagatc ggcccgcgca
     1441 tggccgagtt gagcggttcc cggctggccg cgcagcaaca gatggaaggc ctcctggcgc
     1501 cgcaccggcc caaggagccc gcgtggttcc tggccaccgt cggcgtctcg cccgaccacc
     1561 agggcaaggg tctgggcagc gccgtcgtgc tccccggagt ggaggcggcc gagcgcgccg
     1621 gggtgcccgc cttcctggag acctccgcgc cccgcaacct ccccttctac gagcggctcg
     1681 gcttcaccgt caccgccgac gtcgaggtgc ccgaaggacc gcgcacctgg tgcatgaccc
     1741 gcaagcccgg tgccggatcg ggagagggca gaggaagtct gctaacatgc ggtgacgtcg
     1801 aggagaatcc tggcccaccg gtcgccacca tggtgagcaa gggcgaggag gataacatgg
     1861 ccatcatcaa ggagttcatg cgcttcaagg tgcacatgga gggctccgtg aacggccacg
     1921 agttcgagat cgagggcgag ggcgagggcc gcccctacga gggcacccag accgccaagc
     1981 tgaaggtgac caagggtggc cccctgccct tcgcctggga catcctgtcc cctcagttca
     2041 tgtacggctc caaggcctac gtgaagcacc ccgccgacat ccccgactac ttgaagctgt
     2101 ccttccccga gggcttcaag tgggagcgcg tgatgaactt cgaggacggc ggcgtggtga
     2161 ccgtgaccca ggactcctcc ctgcaggacg gcgagttcat ctacaaggtg aagctgcgcg
     2221 gcaccaactt cccctccgac ggccccgtaa tgcagaagaa gaccatgggc tgggaggcct
     2281 cctccgagcg gatgtacccc gaggacggcg ccctgaaggg cgagatcaag cagaggctga
     2341 agctgaagga cggcggccac tacgacgctg aggtcaagac cacctacaag gccaagaagc
     2401 ccgtgcagct gcccggcgcc tacaacgtca acatcaagtt ggacatcacc tcccacaacg
     2461 aggactacac catcgtggaa cagtacgaac gcgccgaggg ccgccactcc accggcggca
     2521 tggacgagct gtacaaggga gctgatccaa aaaagaagag aaaggtatag ttaattaagt
     2581 cgagggacct aacatgttta agggttccgg ttccactagg tacaattcga tatcaagctt
     2641 atcgataatc aacctctgga ttacaaaatt tgtgaaagat tgactggtat tcttaactat
     2701 gttgctcctt ttacgctatg tggatacgct gctttaatgc ctttgtatca tgctattgct
     2761 tcccgtatgg ctttcatttt ctcctccttg tataaatcct ggttgctgtc tctttatgag
     2821 gagttgtggc ccgttgtcag gcaacgtggc gtggtgtgca ctgtgtttgc tgacgcaacc
     2881 cccactggtt ggggcattgc caccacctgt cagctccttt ccgggacttt cgctttcccc
     2941 ctccctattg ccacggcgga actcatcgcc gcctgccttg cccgctgctg gacaggggct
     3001 cggctgttgg gcactgacaa ttccgtggtg ttgtcgggga aatcatcgtc ctttccttgg
     3061 ctgctcgcct gtgttgccac ctggattctg cgcgggacgt ccttctgcta cgtcccttcg
     3121 gccctcaatc cagcggacct tccttcccgc ggcctgctgc cggctctgcg gcctcttccg
     3181 cgtcttcgcc ttcgccctca gacgagtcgg atctcccttt gggccgcctc cccgcatcga
     3241 taccgtcgac ctcgatcgag acctagaaaa acatggagca atcacaagta gcaatacagc
     3301 agctaccaat gctgattgtg cctggctaga agcacaagag gaggaggagg tgggttttcc
     3361 agtcacacct caggtacctt taagaccaat gacttacaag gcagctgtag atcttagcca
     3421 ctttttaaaa gaaaaggggg gactggaagg gctaattcac tcccaacgaa gacaagatat
     3481 ccttgatctg tggatctacc acacacaagg ctacttccct gattggcaga actacacacc
     3541 agggccaggg atcagatatc cactgacctt tggatggtgc tacaagctag taccagttga
     3601 gcaagagaag gtagaagaag ccaatgaagg agagaacacc cgcttgttac accctgtgag
     3661 cctgcatggg atggatgacc cggagagaga agtattagag tggaggtttg acagccgcct
     3721 agcatttcat cacatggccc gagagctgca tccggactgt actgggtctc tctggttaga
     3781 ccagatctga gcctgggagc tctctggcta actagggaac ccactgctta agcctcaata
     3841 aagcttgcct tgagtgcttc aagtagtgtg tgcccgtctg ttgtgtgact ctggtaacta
     3901 gagatccctc agaccctttt agtcagtgtg gaaaatctct agcagcatgt gagcaaaagg
     3961 ccagcaaaag gccaggaacc gtaaaaaggc cgcgttgctg gcgtttttcc ataggctccg
     4021 cccccctgac gagcatcaca aaaatcgacg ctcaagtcag aggtggcgaa acccgacagg
     4081 actataaaga taccaggcgt ttccccctgg aagctccctc gtgcgctctc ctgttccgac
     4141 cctgccgctt accggatacc tgtccgcctt tctcccttcg ggaagcgtgg cgctttctca
     4201 tagctcacgc tgtaggtatc tcagttcggt gtaggtcgtt cgctccaagc tgggctgtgt
     4261 gcacgaaccc cccgttcagc ccgaccgctg cgccttatcc ggtaactatc gtcttgagtc
     4321 caacccggta agacacgact tatcgccact ggcagcagcc actggtaaca ggattagcag
     4381 agcgaggtat gtaggcggtg ctacagagtt cttgaagtgg tggcctaact acggctacac
     4441 tagaagaaca gtatttggta tctgcgctct gctgaagcca gttaccttcg gaaaaagagt
     4501 tggtagctct tgatccggca aacaaaccac cgctggtagc ggtggttttt ttgtttgcaa
     4561 gcagcagatt acgcgcagaa aaaaaggatc tcaagaagat cctttgatct tttctacggg
     4621 gtctgacgct cagtggaacg aaaactcacg ttaagggatt ttggtcatga gattatcaaa
     4681 aaggatcttc acctagatcc ttttaaatta aaaatgaagt tttaaatcaa tctaaagtat
     4741 atatgagtaa acttggtctg acagttacca atgcttaatc agtgaggcac ctatctcagc
     4801 gatctgtcta tttcgttcat ccatagttgc ctgactcccc gtcgtgtaga taactacgat
     4861 acgggagggc ttaccatctg gccccagtgc tgcaatgata ccgcgagacc cacgctcacc
     4921 ggctccagat ttatcagcaa taaaccagcc agccggaagg gccgagcgca gaagtggtcc
     4981 tgcaacttta tccgcctcca tccagtctat taattgttgc cgggaagcta gagtaagtag
     5041 ttcgccagtt aatagtttgc gcaacgttgt tgccattgct acaggcatcg tggtgtcacg
     5101 ctcgtcgttt ggtatggctt cattcagctc cggttcccaa cgatcaaggc gagttacatg
     5161 atcccccatg ttgtgcaaaa aagcggttag ctccttcggt cctccgatcg ttgtcagaag
     5221 taagttggcc gcagtgttat cactcatggt tatggcagca ctgcataatt ctcttactgt
     5281 catgccatcc gtaagatgct tttctgtgac tggtgagtac tcaaccaagt cattctgaga
     5341 atagtgtatg cggcgaccga gttgctcttg cccggcgtca atacgggata ataccgcgcc
     5401 acatagcaga actttaaaag tgctcatcat tggaaaacgt tcttcggggc gaaaactctc
     5461 aaggatctta ccgctgttga gatccagttc gatgtaaccc actcgtgcac ccaactgatc
     5521 ttcagcatct tttactttca ccagcgtttc tgggtgagca aaaacaggaa ggcaaaatgc
     5581 cgcaaaaaag ggaataaggg cgacacggaa atgttgaata ctcatactct tcctttttca
     5641 atattattga agcatttatc agggttattg tctcatgagc ggatacatat ttgaatgtat
     5701 ttagaaaaat aaacaaatag gggttccgcg cacatttccc cgaaaagtgc cacctgacgt
     5761 cgacggatcg ggagatctcc cgatccccta tggtgcactc tcagtacaat ctgctctgat
     5821 gccgcatagt taagccagta tctgctccct gcttgtgtgt tggaggtcgc tgagtagtgc
     5881 gcgagcaaaa tttaagctac aacaaggcaa ggcttgaccg acaattgcat gaagaatctg
     5941 cttagggtta ggcgttttgc gctgcttcgc gatgtacggg ccagatatac gcgttgacat
     6001 tgattattga ctagttatta atagtaatca attacggggt cattagttca tagcccatat
     6061 atggagttcc gcgttacata acttacggta aatggcccgc ctggctgacc gcccaacgac
     6121 ccccgcccat tgacgtcaat aatgacgtat gttcccatag taacgccaat agggactttc
     6181 cattgacgtc aatgggtgga gtatttacgg taaactgccc acttggcagt acatcaagtg
     6241 tatcatatgc caagtacgcc ccctattgac gtcaatgacg gtaaatggcc cgcctggcat
     6301 tatgcccagt acatgacctt atgggacttt cctacttggc agtacatcta cgtattagtc
     6361 atcgctatta ccatggtgat gcggttttgg cagtacatca atgggcgtgg atagcggttt
     6421 gactcacggg gatttccaag tctccacccc attgacgtca atgggagttt gttttggcac
     6481 caaaatcaac gggactttcc aaaatgtcgt aacaactccg ccccattgac gcaaatgggc
     6541 ggtaggcgtg tacggtggga ggtctatata agcagcgcgt tttgcctgta ctgggtctct
     6601 ctggttagac cagatctgag cctgggagct ctctggctaa ctagggaacc cactgcttaa
     6661 gcctcaataa agcttgcctt gagtgcttca agtagtgtgt gcccgtctgt tgtgtgactc
     6721 tggtaactag agatccctca gaccctttta gtcagtgtgg aaaatctcta gcagtggcgc
     6781 ccgaacaggg acttgaaagc gaaagggaaa ccagaggagc tctctcgacg caggactcgg
     6841 cttgctgaag cgcgcacggc aagaggcgag gggcggcgac tggtgagtac gccaaaaatt
     6901 ttgactagcg gaggctagaa ggagagagat gggtgcgaga gcgtcagtat taagcggggg
     6961 agaattagat cgcgatggga aaaaattcgg ttaaggccag ggggaaagaa aaaatataaa
     7021 ttaaaacata tagtatgggc aagcagggag ctagaacgat tcgcagttaa tcctggcctg
     7081 ttagaaacat cagaaggctg tagacaaata ctgggacagc tacaaccatc ccttcagaca
     7141 ggatcagaag aacttagatc attatataat acagtagcaa ccctctattg tgtgcatcaa
     7201 aggatagaga taaaagacac caaggaagct ttagacaaga tagaggaaga gcaaaacaaa
     7261 agtaagacca ccgcacagca agcggccggc cgctgatctt cagacctgga ggaggagata
     7321 tgagggacaa ttggagaagt gaattatata aatataaagt agtaaaaatt gaaccattag
     7381 gagtagcacc caccaaggca aagagaagag tggtgcagag agaaaaaaga gcagtgggaa
     7441 taggagcttt gttccttggg ttcttgggag cagcaggaag cactatgggc gcagcgtcaa
     7501 tgacgctgac ggtacaggcc agacaattat tgtctggtat agtgcagcag cagaacaatt
     7561 tgctgagggc tattgaggcg caacagcatc tgttgcaact cacagtctgg ggcatcaagc
     7621 agctccaggc aagaatcctg gctgtggaaa gatacctaaa ggatcaacag ctcctgggga
     7681 tttggggttg ctctggaaaa ctcatttgca ccactgctgt gccttggaat gctagttgga
     7741 gtaataaatc tctggaacag atttggaatc acacgacctg gatggagtgg gacagagaaa
     7801 ttaacaatta cacaagctta atacactcct taattgaaga atcgcaaaac cagcaagaaa
     7861 agaatgaaca agaattattg gaattagata aatgggcaag tttgtggaat tggtttaaca
     7921 taacaaattg gctgtggtat ataaaattat tcataatgat agtaggaggc ttggtaggtt
     7981 taagaatagt ttttgctgta ctttctatag tgaatagagt taggcaggga tattcaccat
     8041 tatcgtttca gacccacctc ccaaccccga ggggacccga caggcccgaa ggaatagaag
     8101 aagaaggtgg agagagagac agagacagat ccattcgatt agtgaacgga tcggcactgc
     8161 gtgcgccaat tctgcagaca aatggcagta ttcatccaca attttaaaag aaaagggggg
     8221 attggggggt acagtgcagg ggaaagaata gtagacataa tagcaacaga catacaaact
     8281 aaagaattac aaaaacaaat tacaaaaatt caaaattttc gggtttatta cagggacagc
     8341 agagatccag tttggttagt accgggcccg ctctagagat ccgacgcgcc atctctaggc
     8401 ccgcgccggc cccctcgcac ggacttgtgg gagaagctcg gctactcccc tgccccggtt
     8461 aatttgcata taatatttcc tagtaactat agaggcttaa tgtgcgataa aagacagata
     8521 atctgttctt tttaatacta gctacatttt acatgatagg cttggatttc tataacttcg
     8581 tatagcatac attatacgaa gttataaaca gcacaaaagg aaactcaccc taactgtaaa
     8641 gtaattgtgt gttttgagac tataagtatc ccttggagaa ccaccttgtt ggagacgcag
     8701 gtgtcactgg cgctgttggt gccggcacct gacacagcag aggatgcaga gcccaaggaa
     8761 acccagaaag ccgaaggagc cacccagcac gcaagccagg aacactgtct ggttcacccc
     8821 tctggataaa acctggatcc tggagtccat cttgacctta tcaccttcac tcagtagaca
     8881 ctgccacagc cctgtctcag gggccaccac ttgaactact ttctgctcct cagagaccct
     8941 ggcctcctgg ttctcctgct tcagggtcag tctcatcttg ggagaggtag gtcccatcac
     9001 ctcacaggtc aaagtattgt tgagctgagc cactttcatc accaccaggt tcacttcctg
     9061 atgcagtgtc cctttgtcca gagtcagagt caggttgcca gaaccagcaa actgaagcga
     9121 gacctggggt atcttgaggg tgagtgggag cgtttccttc agctggagct tgaggtcttt
     9181 ggtggacttt tgtacggaca cctctttgtt ctttatggag aaggagatcc agggctggaa
     9241 gaaagaatcc ttctctgcct tccacatcag ctctccccac ccgttttcct ctgcaaagtt
     9301 gagtgggaag gagaactccg ctgactctcc ctcactctta taggccgtga tagctgtgct
     9361 ctgaaaaccc agcactgaga gtgtcatgcc gaaccagttc tttttctggt ccagggtcac
     9421 ggtgcagttc cagaagtcgc tgtcctgaac ccttaggttg gacatggaga gaactttgga
     9481 accactgaca actttaccct ttttgtgttt gcactctgtc aaggggttag agaccttaga
     9541 gttgctatcc aaggtcaggg tcaggctctg cccttgcaac aggctggtac ccggactgaa
     9601 ggtcactttg aacacccaca actccacctc ctctttcctg ttctccagct cacagatata
     9661 agtctgagag tcttccatct taagtttatt gatgatgaga ggaaacgatc ctttctccca
     9721 tgcccctttt ttggaatcaa aacgatcaaa ctgcgaaggc gaacctcctc taattaatac
     9781 acctttgcca tgctgcccca gaatcttcct ctggtcagag aacttccagg tgaagactgt
     9841 gatcttcttc tgggaactct cgcagggcag ttctgctgat tccccttcct tccccagcac
     9901 cagcgtcttc ccttgagtga cagctaggag ttgtgacagc tgcagcagca gcagcagcaa
     9961 gcgcctaaga gagatggctc ggcacatgtc acctgcgatc gtttaagagc tatgctggaa
    10021 acagcatagc aagtttaaat aaggctagtc cgttatcaac ttgaaaaagt ggcaccgagt
    10081 cggtgctttt tttgcggccg cgtggataac cgtattaccg ccatgcagcc taatctcatt
    10141 cgatactcat accagatcgg aagagcacac gtctgaactc cagtcactag gtcttgaaag
    10201 gagtgggaat tggctccggt gcccgtcagt gggcagagcg cacatcgccc acagtccccg
    10261 agaagttggg gggaggggtc ggcaattgat ccggtgccta gagaaggtgg cgcggggtaa
    10321 actgggaaag tgatgtcgtg tactggctcc gcctttttcc cgagggtggg ggagaaccgt
    10381 atataagtgc agtagtcgcc gtgaacgttc tttttcgcaa cgggtttgcc gccagaacac
    10441 aggaccggtt ctagagcgct gccaccatgg acaagaagta cagcatcggc ctggacatcg
    10501 gcaccaactc tgtgggctgg gccgtgatca ccgacgagta caaggtgccc agcaagaaat
    10561 tcaaggtgct gggcaacacc gaccggcaca gcatcaagaa gaacctgatc ggagccctgc
    10621 tgttcgacag cggcgaaaca gccgaggcca cccggctgaa gagaaccgcc agaagaagat
    10681 acaccagacg gaagaaccgg atctgctatc tgcaagagat cttcagcaac gagatggcca
    10741 aggtggacga cagcttcttc cacagactgg aagagtcctt cctggtggaa gaggataaga
    10801 agcacgagcg gcaccccatc ttcggcaaca tcgtggacga ggtggcctac cacgagaagt
    10861 accccaccat ctaccacctg agaaagaaac tggtggacag caccgacaag gccgacctgc
    10921 ggctgatcta tctggccctg gcccacatga tcaagttccg gggccacttc ctgatcgagg
    10981 gcgacctgaa ccccgacaac agcgacgtgg acaagctgtt catccagctg gtgcagacct
    11041 acaaccagct gttcgaggaa aaccccatca acgccagcgg cgtggacgcc aaggccatcc
    11101 tgtctgccag actgagcaag agcagacggc tggaaaatct gatcgcccag ctgcccggcg
    11161 agaagaagaa tggcctgttc ggaaacctga ttgccctgag cctgggcctg acccccaact
    11221 tcaagagcaa cttcgacctg gccgaggatg ccaaactgca gctgagcaag gacacctacg
    11281 acgacgacct ggacaacctg ctggcccaga tcggcgacca gtacgccgac ctgtttctgg
    11341 ccgccaagaa cctgtccgac gccatcctgc tgagcgacat cctgagagtg aacaccgaga
    11401 tcaccaaggc ccccctgagc gcctctatga tcaagagata cgacgagcac caccaggacc
    11461 tgaccctgct gaaagctctc gtgcggcagc agctgcctga gaagtacaaa gagattttct
    11521 tcgaccagag caagaacggc tacgccggct acattgacgg cggagccagc caggaagagt
    11581 tctacaagtt catcaagccc atcctggaaa agatggacgg caccgaggaa ctgctcgtga
    11641 agctgaacag agaggacctg ctgcggaagc agcggacctt cgacaacggc agcatccccc
    11701 accagatcca cctgggagag ctgcacgcca ttctgcggcg gcaggaagat ttttacccat
    11761 tcctgaagga caaccgggaa aagatcgaga agatcctgac cttccgcatc ccctactacg
    11821 tgggccctct ggccagggga aacagcagat tcgcctggat gaccagaaag agcgaggaaa
    11881 ccatcacccc ctggaacttc gaggaagtgg tggacaaggg cgcttccgcc cagagcttca
    11941 tcgagcggat gaccaacttc gataagaacc tgcccaacga gaaggtgctg cccaagcaca
    12001 gcctgctgta cgagtacttc accgtgtata acgagctgac caaagtgaaa tacgtgaccg
    12061 agggaatgag aaagcccgcc ttcctgagcg gcgagcagaa aaaggccatc gtggacctgc
    12121 tgttcaagac caaccggaaa gtgaccgtga agcagctgaa agaggactac ttcaagaaaa
    12181 tcgagtgctt cgactccgtg gaaatctccg gcgtggaaga tcggttcaac gcctccctgg
    12241 gcacatacca cgatctgctg aaaattatca aggacaagga cttcctggac aatgaggaaa
    12301 acgaggacat tctggaagat atcgtgctga ccctgacact gtttgaggac agagagatga
    12361 tcgaggaacg gctgaaaacc tatgcccacc tgttcgacga caaagtgatg aagcagctga
    12421 agcggcggag atacaccggc tggggcaggc tgagccggaa gctgatcaac ggcatccggg
    12481 acaagcagtc cggcaagaca atcctggatt tcctgaagtc cgacggcttc gccaacagaa
    12541 acttcatgca gctgatccac gacgacagcc tgacctttaa agaggacatc cagaaagccc
    12601 aggtgtccgg ccagggcgat agcctgcacg agcacattgc caatctggcc ggcagccccg
    12661 ccattaagaa gggcatcctg cagacagtga aggtggtgga cgagctcgtg aaagtgatgg
    12721 gccggcacaa gcccgagaac atcgtgatcg aaatggccag agagaaccag accacccaga
    12781 agggacagaa gaacagccgc gagagaatga agcggatcga agagggcatc aaagagctgg
    12841 gcagccagat cctgaaagaa caccccgtgg aaaacaccca gctgcagaac gagaagctgt
    12901 acctgtacta cctgcagaat gggcgggata tgtacgtgga ccaggaactg gacatcaacc
    12961 ggctgtccga ctacgatgtg gaccatatcg tgcctcagag ctttctgaag gacgactcca
    13021 tcgacaacaa ggtgctgacc agaagcgaca agaaccgggg caagagcgac aacgtgccct
    13081 ccgaagaggt cgtgaagaag atgaagaact actggcggca gctgctgaac gccaagctga
    13141 ttacccagag aaagttcgac aatctgacca aggccgagag aggcggcctg agcgaactgg
    13201 ataaggccgg cttcatcaag agacagctgg tggaaacccg gcagatcaca aagcacgtgg
    13261 cacagatcct ggactcccgg atgaacacta agtacgacga gaatgacaag ctgatccggg
    13321 aagtgaaagt gatcaccctg aagtccaagc tggtgtccga tttccggaag gatttccagt
    13381 tttacaaagt gcgcgagatc aacaactacc accacgccca cgacgcctac ctgaacgccg
    13441 tcgtgggaac cgccctgatc aaaaagtacc ctaagctgga aagcgagttc gtgtacggcg
    13501 actacaaggt gtacgacgtg cggaagatga tcgccaagag cgagcagg
//
