LOCUS       sequence-487498-       14585 bp    DNA     circular SYN 30-SEP-2025
DEFINITION  Constitutive active CRISPRgenee construct with a blasticidin 
            resistance.
ACCESSION   .
VERSION     .
KEYWORDS    .
SOURCE      synthetic DNA construct
  ORGANISM  synthetic DNA construct
REFERENCE   1  (bases 1 to 14585)
  AUTHORS   Stadager J, Bernardini C, Hartmann L, May H, Wiepcke J, Kuban M, 
            Najafova Z, Johnsen SA, Legewie S, Traube FR, Jude J, Rathert P
  TITLE     CRISPR GENome and epigenome engineering improves loss-of-function 
            genetic-screening approaches.
  JOURNAL   Cell Rep Methods. 2025 Jun 16;5(6):101078. doi: 
            10.1016/j.crmeth.2025.101078. Epub 2025 Jun 10.
  PUBMED    40499551
REFERENCE   2  (bases 1 to 14585)
  AUTHORS   .
  TITLE     Direct Submission
  JOURNAL   Exported Sep 30, 2025 from SnapGene Server 8.0.1
            https://www.snapgene.com
COMMENT     SGRef: number: 1; type: "Journal Article"; journalName: "Cell Rep 
            Methods."; date: "2025-06-16"; volume: "5(6):101078. doi"; pages: " 
            10.1016/j.crmeth.2025.101078. Epub 2025 Jun 10"
COMMENT     Sequence Label: EF1a-ZIM3-Cas9-P2A-GFP-PGK-Blasti
FEATURES             Location/Qualifiers
     source          1..14585
                     /mol_type="other DNA"
                     /organism="synthetic DNA construct"
     CDS             complement(join(340..1131,1132..1200))
                     /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     963..982
                     /label=Amp-R
                     /note="Ampicillin resistance gene, reverse primer"
     promoter        complement(1201..1305)
                     /gene="bla"
                     /label=AmpR promoter
     primer_bind     complement(1380..1399)
                     /label=pRS-marker
                     /note="pRS vectors, use to sequence yeast selectable 
                     marker"
     enhancer        1571..1950
                     /label=CMV enhancer
                     /note="human cytomegalovirus immediate early enhancer"
     promoter        1952..2150
                     /label=CMV promoter
                     /note="human cytomegalovirus (CMV) immediate early 
                     promoter"
     primer_bind     2105..2125
                     /label=CMV-F
                     /note="Human CMV immediate early promoter, forward primer"
     LTR             2168..2348
                     /label=5' LTR (truncated)
                     /note="truncated 5' long terminal repeat (LTR) from HIV-1"
     misc_feature    2395..2520
                     /label=HIV-1 Psi
                     /note="packaging signal of human immunodeficiency virus 
                     type 1"
     misc_feature    3013..3246
                     /label=RRE
                     /note="The Rev response element (RRE) of HIV-1 allows for 
                     Rev-dependent mRNA export from the nucleus to the 
                     cytoplasm."
     CDS             3431..3475
                     /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    3773..3890
                     /label=cPPT/CTS
                     /note="central polypurine tract and central termination 
                     sequence of HIV-1"
     promoter        3983..4194
                     /label=EF-1-alpha core promoter
                     /note="core promoter for human elongation factor 
                     EF-1-alpha"
     protein_bind    complement(4510..4534)
                     /gene="mutant version of attB"
                     /label=attB2
                     /bound_moiety="BP Clonase(TM)"
                     /note="recombination site for the Gateway(R) BP reaction"
     CDS             4554..4574
                     /codon_start=1
                     /product="nuclear localization signal of SV40 (simian virus
                     40) large T antigen"
                     /label=SV40 NLS
                     /translation="PKKKRKV"
     regulatory      4629..4638
                     /label=Kozak sequence
                     /note="vertebrate consensus sequence for strong initiation 
                     of translation (Kozak, 1987)"
                     /regulatory_class="other"
     CDS             4635..8738
                     /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             4635..8738
                     /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             8739..8786
                     /codon_start=1
                     /product="bipartite nuclear localization signal from 
                     nucleoplasmin"
                     /label=nucleoplasmin NLS
                     /translation="KRPAATKKAGQAKKKK"
     CDS             8787..8810
                     /codon_start=1
                     /product="FLAG(R) epitope tag, followed by an enterokinase 
                     cleavage site"
                     /label=FLAG
                     /translation="DYKDDDDK"
     CDS             8826..8882
                     /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             join(8883..8885,8886..8888,8889..9602)
                     /codon_start=1
                     /product="the original enhanced GFP (Yang et al., 1996)"
                     /label=EGFP
                     /note="mammalian codon-optimized"
                     /translation="MVSKGEELFTGVVPILVELDGDVNGHKFSVSGEGEGDATYGKLTL
                     KFICTTGKLPVPWPTLVTTLTYGVQCFSRYPDHMKQHDFFKSAMPEGYVQERTIFFKDD
                     GNYKTRAEVKFEGDTLVNRIELKGIDFKEDGNILGHKLEYNYNSHNVYIMADKQKNGIK
                     ANFKIRHNIEDGSVQLADHYQQNTPIGDGPVLLPDNHYLSTQSALSKDPNEKRDHMVLL
                     EFVTAAGITLGMDELYK"
     primer_bind     complement(9189..9208)
                     /label=EXFP-R
                     /note="For distinguishing EGFP variants, reverse primer"
     primer_bind     9536..9557
                     /label=EGFP-C
                     /note="EGFP, forward primer"
     promoter        9621..10120
                     /label=PGK promoter
                     /note="mouse phosphoglycerate kinase 1 promoter"
     primer_bind     complement(9656..9679)
                     /label=MSCV-rev
                     /note="Murine stem cell virus, reverse primer"
     primer_bind     10053..10072
                     /label=mPGK-F
                     /note="Mouse PGK promoter, forward primer"
     misc_feature    10610..11198
                     /label=WPRE
                     /note="woodchuck hepatitis virus posttranscriptional 
                     regulatory element"
     primer_bind     complement(10663..10683)
                     /label=WPRE-R
                     /note="WPRE, reverse primer"
     CDS             complement(11081..11092)
                     /codon_start=1
                     /product="Factor Xa recognition and cleavage site"
                     /label=Factor Xa site
                     /translation="IEGR"
     primer_bind     complement(11201..11217)
                     /label=KS primer
                     /note="common sequencing primer, one of multiple similar 
                     variants"
     primer_bind     complement(11202..11218)
                     /label=pBluescriptKS
                     /note="For pBluescript vector"
     LTR             11723..11903
                     /label=5' LTR (truncated)
                     /note="truncated 5' long terminal repeat (LTR) from HIV-1"
     primer_bind     complement(11929..11946)
                     /label=BGH-rev
                     /note="Bovine growth hormone terminator, reverse primer. 
                     Also called BGH reverse"
     polyA_signal    11935..12159
                     /label=bGH poly(A) signal
                     /note="bovine growth hormone polyadenylation signal"
     rep_origin      12205..12633
                     /direction=RIGHT
                     /label=f1 ori
                     /note="f1 bacteriophage origin of replication; arrow 
                     indicates direction of (+) strand synthesis"
     primer_bind     complement(12292..12311)
                     /label=F1ori-R
                     /note="F1 origin, reverse primer"
     primer_bind     12502..12523
                     /label=F1ori-F
                     /note="F1 origin, forward primer"
     primer_bind     complement(12642..12662)
                     /label=pBABE 3'
                     /note="SV40 enhancer, reverse primer for pBABE vectors"
     promoter        12647..12976
                     /label=SV40 promoter
                     /note="SV40 enhancer and early promoter"
     rep_origin      12827..12962
                     /label=SV40 ori
                     /note="SV40 origin of replication"
     primer_bind     12889..12908
                     /label=SV40pro-F
                     /note="SV40 promoter/origin, forward primer"
     promoter        13024..13071
                     /label=EM7 promoter
                     /note="synthetic bacterial promoter "
     CDS             13090..13464
                     /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    13594..13715
                     /label=SV40 poly(A) signal
                     /note="SV40 polyadenylation signal"
     primer_bind     complement(13631..13650)
                     /label=SV40pA-R
                     /note="SV40 polyA, reverse primer"
     primer_bind     13685..13704
                     /label=EBV-rev
                     /note="SV40 polyA terminator, reverse primer"
     primer_bind     complement(13764..13780)
                     /label=M13 rev
                     /note="common sequencing primer, one of multiple similar 
                     variants"
     primer_bind     complement(13764..13780)
                     /label=M13 Reverse
                     /note="In lacZ gene. Also called M13-rev"
     primer_bind     complement(13777..13799)
                     /label=M13/pUC Reverse
                     /note="In lacZ gene"
     protein_bind    13788..13804
                     /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(13812..13818,13819..13836,13837..13842))
                     /label=lac promoter
                     /note="promoter for the E. coli lac operon"
     protein_bind    13857..13878
                     /label=CAP binding site
                     /bound_moiety="E. coli catabolite activator protein"
                     /note="CAP binding activates transcription in the presence 
                     of cAMP."
     primer_bind     complement(13995..14012)
                     /label=L4440
                     /note="L4440 vector, forward primer"
     rep_origin      complement(join(14166..14585,1..169))
                     /direction=LEFT
                     /label=ori
                     /note="high-copy-number ColE1/pMB1/pBR322/pUC origin of 
                     replication"
     primer_bind     complement(14246..14265)
                     /label=pBR322ori-F
                     /note="pBR322 origin, forward primer"
ORIGIN
        1 taactacggc tacactagaa gaacagtatt tggtatctgc gctctgctga agccagttac
       61 cttcggaaaa agagttggta gctcttgatc cggcaaacaa accaccgctg gtagcggtgg
      121 tttttttgtt tgcaagcagc agattacgcg cagaaaaaaa ggatctcaag aagatccttt
      181 gatcttttct acggggtctg acgctcagtg gaacgaaaac tcacgttaag ggattttggt
      241 catgagatta tcaaaaagga tcttcaccta gatcctttta aattaaaaat gaagttttaa
      301 atcaatctaa agtatatatg agtaaacttg gtctgacagt taccaatgct taatcagtga
      361 ggcacctatc tcagcgatct gtctatttcg ttcatccata gttgcctgac tccccgtcgt
      421 gtagataact acgatacggg agggcttacc atctggcccc agtgctgcaa tgataccgcg
      481 agacccacgc tcaccggctc cagatttatc agcaataaac cagccagccg gaagggccga
      541 gcgcagaagt ggtcctgcaa ctttatccgc ctccatccag tctattaatt gttgccggga
      601 agctagagta agtagttcgc cagttaatag tttgcgcaac gttgttgcca ttgctacagg
      661 catcgtggtg tcacgctcgt cgtttggtat ggcttcattc agctccggtt cccaacgatc
      721 aaggcgagtt acatgatccc ccatgttgtg caaaaaagcg gttagctcct tcggtcctcc
      781 gatcgttgtc agaagtaagt tggccgcagt gttatcactc atggttatgg cagcactgca
      841 taattctctt actgtcatgc catccgtaag atgcttttct gtgactggtg agtactcaac
      901 caagtcattc tgagaatagt gtatgcggcg accgagttgc tcttgcccgg cgtcaatacg
      961 ggataatacc gcgccacata gcagaacttt aaaagtgctc atcattggaa aacgttcttc
     1021 ggggcgaaaa ctctcaagga tcttaccgct gttgagatcc agttcgatgt aacccactcg
     1081 tgcacccaac tgatcttcag catcttttac tttcaccagc gtttctgggt gagcaaaaac
     1141 aggaaggcaa aatgccgcaa aaaagggaat aagggcgaca cggaaatgtt gaatactcat
     1201 actcttcctt tttcaatatt attgaagcat ttatcagggt tattgtctca tgagcggata
     1261 catatttgaa tgtatttaga aaaataaaca aataggggtt ccgcgcacat ttccccgaaa
     1321 agtgccacct gacgtcgacg gatcgggaga tctcccgatc ccctatggtg cactctcagt
     1381 acaatctgct ctgatgccgc atagttaagc cagtatctgc tccctgcttg tgtgttggag
     1441 gtcgctgagt agtgcgcgag caaaatttaa gctacaacaa ggcaaggctt gaccgacaat
     1501 tgcatgaaga atctgcttag ggttaggcgt tttgcgctgc ttcgcgatgt acgggccaga
     1561 tatacgcgtt gacattgatt attgactagt tattaatagt aatcaattac ggggtcatta
     1621 gttcatagcc catatatgga gttccgcgtt acataactta cggtaaatgg cccgcctggc
     1681 tgaccgccca acgacccccg cccattgacg tcaataatga cgtatgttcc catagtaacg
     1741 ccaataggga ctttccattg acgtcaatgg gtggagtatt tacggtaaac tgcccacttg
     1801 gcagtacatc aagtgtatca tatgccaagt acgcccccta ttgacgtcaa tgacggtaaa
     1861 tggcccgcct ggcattatgc ccagtacatg accttatggg actttcctac ttggcagtac
     1921 atctacgtat tagtcatcgc tattaccatg gtgatgcggt tttggcagta catcaatggg
     1981 cgtggatagc ggtttgactc acggggattt ccaagtctcc accccattga cgtcaatggg
     2041 agtttgtttt ggcaccaaaa tcaacgggac tttccaaaat gtcgtaacaa ctccgcccca
     2101 ttgacgcaaa tgggcggtag gcgtgtacgg tgggaggtct atataagcag cgcgttttgc
     2161 ctgtactggg tctctctggt tagaccagat ctgagcctgg gagctctctg gctaactagg
     2221 gaacccactg cttaagcctc aataaagctt gccttgagtg cttcaagtag tgtgtgcccg
     2281 tctgttgtgt gactctggta actagagatc cctcagaccc ttttagtcag tgtggaaaat
     2341 ctctagcagt ggcgcccgaa cagggacttg aaagcgaaag ggaaaccaga ggagctctct
     2401 cgacgcagga ctcggcttgc tgaagcgcgc acggcaagag gcgaggggcg gcgactggtg
     2461 agtacgccaa aaattttgac tagcggaggc tagaaggaga gagatgggtg cgagagcgtc
     2521 agtattaagc gggggagaat tagatcgcga tgggaaaaaa ttcggttaag gccaggggga
     2581 aagaaaaaat ataaattaaa acatatagta tgggcaagca gggagctaga acgattcgca
     2641 gttaatcctg gcctgttaga aacatcagaa ggctgtagac aaatactggg acagctacaa
     2701 ccatcccttc agacaggatc agaagaactt agatcattat ataatacagt agcaaccctc
     2761 tattgtgtgc atcaaaggat agagataaaa gacaccaagg aagctttaga caagatagag
     2821 gaagagcaaa acaaaagtaa gaccaccgca cagcaagcgg ccgctgatct tcagacctgg
     2881 aggaggagat atgagggaca attggagaag tgaattatat aaatataaag tagtaaaaat
     2941 tgaaccatta ggagtagcac ccaccaaggc aaagagaaga gtggtgcaga gagaaaaaag
     3001 agcagtggga ataggagctt tgttccttgg gttcttggga gcagcaggaa gcactatggg
     3061 cgcagcgtca atgacgctga cggtacaggc cagacaatta ttgtctggta tagtgcagca
     3121 gcagaacaat ttgctgaggg ctattgaggc gcaacagcat ctgttgcaac tcacagtctg
     3181 gggcatcaag cagctccagg caagaatcct ggctgtggaa agatacctaa aggatcaaca
     3241 gctcctgggg atttggggtt gctctggaaa actcatttgc accactgctg tgccttggaa
     3301 tgctagttgg agtaataaat ctctggaaca gatttggaat cacacgacct ggatggagtg
     3361 ggacagagaa attaacaatt acacaagctt aatacactcc ttaattgaag aatcgcaaaa
     3421 ccagcaagaa aagaatgaac aagaattatt ggaattagat aaatgggcaa gtttgtggaa
     3481 ttggtttaac ataacaaatt ggctgtggta tataaaatta ttcataatga tagtaggagg
     3541 cttggtaggt ttaagaatag tttttgctgt actttctata gtgaatagag ttaggcaggg
     3601 atattcacca ttatcgtttc agacccacct cccaaccccg aggggacccg acaggcccga
     3661 aggaatagaa gaagaaggtg gagagagaga cagagacaga tccattcgat tagtgaacgg
     3721 atcggcactg cgtgcgccaa ttctgcagac aaatggcagt attcatccac aattttaaaa
     3781 gaaaaggggg gattgggggg tacagtgcag gggaaagaat agtagacata atagcaacag
     3841 acatacaaac taaagaatta caaaaacaaa ttacaaaaat tcaaaatttt cgggtttatt
     3901 acagggacag cagagatcca gtttggttaa ttagctagct aggtcttgaa aggagtggga
     3961 attggctccg gtgcccgtca gtgggcagag cgcacatcgc ccacagtccc cgagaagttg
     4021 gggggagggg tcggcaattg atccggtgcc tagagaaggt ggcgcggggt aaactgggaa
     4081 agtgatgtcg tgtactggct ccgccttttt cccgagggtg ggggagaacc gtatataagt
     4141 gcagtagtcg ccgtgaacgt tctttttcgc aacgggtttg ccgccagaac acaggaccgg
     4201 ttgccaccat gaacaattcc cagggaagag tgaccttcga ggatgtcact gtgaacttca
     4261 cccaggggga gtggcagcgg ctgaatcccg aacagagaaa cttgtacagg gatgtgatgc
     4321 tggagaatta cagcaacctt gtctctgtgg gacaagggga aaccaccaaa cccgatgtga
     4381 tcttgaggtt ggaacaagga aaggagccat ggttggagga agaggaagtg ctgggaagtg
     4441 gccgtgcaga aaaaaatggg gacattggag ggcagatttg gaagccaaag gatgtgaaag
     4501 agagtctcta cccaactttc ttgtacaaag tggttggtgg atctggaggt tctcccaaga
     4561 agaagcgcaa ggtgggacgc gtctgcagga tatcaagctt gcggtaccgc gggcccggga
     4621 tcagcgctgc caccatggac aagaagtaca gcatcggcct ggacatcggc accaactctg
     4681 tgggctgggc cgtgatcacc gacgagtaca aggtgcccag caagaaattc aaggtgctgg
     4741 gcaacaccga ccggcacagc atcaagaaga acctgatcgg agccctgctg ttcgacagcg
     4801 gcgaaacagc cgaggccacc cggctgaaga gaaccgccag aagaagatac accagacgga
     4861 agaaccggat ctgctatctg caagagatct tcagcaacga gatggccaag gtggacgaca
     4921 gcttcttcca cagactggaa gagtccttcc tggtggaaga ggataagaag cacgagcggc
     4981 accccatctt cggcaacatc gtggacgagg tggcctacca cgagaagtac cccaccatct
     5041 accacctgag aaagaaactg gtggacagca ccgacaaggc cgacctgcgg ctgatctatc
     5101 tggccctggc ccacatgatc aagttccggg gccacttcct gatcgagggc gacctgaacc
     5161 ccgacaacag cgacgtggac aagctgttca tccagctggt gcagacctac aaccagctgt
     5221 tcgaggaaaa ccccatcaac gccagcggcg tggacgccaa ggccatcctg tctgccagac
     5281 tgagcaagag cagacggctg gaaaatctga tcgcccagct gcccggcgag aagaagaatg
     5341 gcctgttcgg aaacctgatt gccctgagcc tgggcctgac ccccaacttc aagagcaact
     5401 tcgacctggc cgaggatgcc aaactgcagc tgagcaagga cacctacgac gacgacctgg
     5461 acaacctgct ggcccagatc ggcgaccagt acgccgacct gtttctggcc gccaagaacc
     5521 tgtccgacgc catcctgctg agcgacatcc tgagagtgaa caccgagatc accaaggccc
     5581 ccctgagcgc ctctatgatc aagagatacg acgagcacca ccaggacctg accctgctga
     5641 aagctctcgt gcggcagcag ctgcctgaga agtacaaaga gattttcttc gaccagagca
     5701 agaacggcta cgccggctac attgacggcg gagccagcca ggaagagttc tacaagttca
     5761 tcaagcccat cctggaaaag atggacggca ccgaggaact gctcgtgaag ctgaacagag
     5821 aggacctgct gcggaagcag cggaccttcg acaacggcag catcccccac cagatccacc
     5881 tgggagagct gcacgccatt ctgcggcggc aggaagattt ttacccattc ctgaaggaca
     5941 accgggaaaa gatcgagaag atcctgacct tccgcatccc ctactacgtg ggccctctgg
     6001 ccaggggaaa cagcagattc gcctggatga ccagaaagag cgaggaaacc atcaccccct
     6061 ggaacttcga ggaagtggtg gacaagggcg cttccgccca gagcttcatc gagcggatga
     6121 ccaacttcga taagaacctg cccaacgaga aggtgctgcc caagcacagc ctgctgtacg
     6181 agtacttcac cgtgtataac gagctgacca aagtgaaata cgtgaccgag ggaatgagaa
     6241 agcccgcctt cctgagcggc gagcagaaaa aggccatcgt ggacctgctg ttcaagacca
     6301 accggaaagt gaccgtgaag cagctgaaag aggactactt caagaaaatc gagtgcttcg
     6361 actccgtgga aatctccggc gtggaagatc ggttcaacgc ctccctgggc acataccacg
     6421 atctgctgaa aattatcaag gacaaggact tcctggacaa tgaggaaaac gaggacattc
     6481 tggaagatat cgtgctgacc ctgacactgt ttgaggacag agagatgatc gaggaacggc
     6541 tgaaaaccta tgcccacctg ttcgacgaca aagtgatgaa gcagctgaag cggcggagat
     6601 acaccggctg gggcaggctg agccggaagc tgatcaacgg catccgggac aagcagtccg
     6661 gcaagacaat cctggatttc ctgaagtccg acggcttcgc caacagaaac ttcatgcagc
     6721 tgatccacga cgacagcctg acctttaaag aggacatcca gaaagcccag gtgtccggcc
     6781 agggcgatag cctgcacgag cacattgcca atctggccgg cagccccgcc attaagaagg
     6841 gcatcctgca gacagtgaag gtggtggacg agctcgtgaa agtgatgggc cggcacaagc
     6901 ccgagaacat cgtgatcgaa atggccagag agaaccagac cacccagaag ggacagaaga
     6961 acagccgcga gagaatgaag cggatcgaag agggcatcaa agagctgggc agccagatcc
     7021 tgaaagaaca ccccgtggaa aacacccagc tgcagaacga gaagctgtac ctgtactacc
     7081 tgcagaatgg gcgggatatg tacgtggacc aggaactgga catcaaccgg ctgtccgact
     7141 acgatgtgga ccatatcgtg cctcagagct ttctgaagga cgactccatc gacaacaagg
     7201 tgctgaccag aagcgacaag aaccggggca agagcgacaa cgtgccctcc gaagaggtcg
     7261 tgaagaagat gaagaactac tggcggcagc tgctgaacgc caagctgatt acccagagaa
     7321 agttcgacaa tctgaccaag gccgagagag gcggcctgag cgaactggat aaggccggct
     7381 tcatcaagag acagctggtg gaaacccggc agatcacaaa gcacgtggca cagatcctgg
     7441 actcccggat gaacactaag tacgacgaga atgacaagct gatccgggaa gtgaaagtga
     7501 tcaccctgaa gtccaagctg gtgtccgatt tccggaagga tttccagttt tacaaagtgc
     7561 gcgagatcaa caactaccac cacgcccacg acgcctacct gaacgccgtc gtgggaaccg
     7621 ccctgatcaa aaagtaccct aagctggaaa gcgagttcgt gtacggcgac tacaaggtgt
     7681 acgacgtgcg gaagatgatc gccaagagcg agcaggaaat cggcaaggct accgccaagt
     7741 acttcttcta cagcaacatc atgaactttt tcaagaccga gattaccctg gccaacggcg
     7801 agatccggaa gcggcctctg atcgagacaa acggcgaaac cggggagatc gtgtgggata
     7861 agggccggga ttttgccacc gtgcggaaag tgctgagcat gccccaagtg aatatcgtga
     7921 aaaagaccga ggtgcagaca ggcggcttca gcaaagagtc tatcctgccc aagaggaaca
     7981 gcgataagct gatcgccaga aagaaggact gggaccctaa gaagtacggc ggcttcgaca
     8041 gccccaccgt ggcctattct gtgctggtgg tggccaaagt ggaaaagggc aagtccaaga
     8101 aactgaagag tgtgaaagag ctactgggga tcaccatcat ggaaagaagc agcttcgaga
     8161 agaatcccat cgactttctg gaagccaagg gctacaaaga agtgaaaaag gacctgatca
     8221 tcaagctgcc taagtactcc ctgttcgagc tggaaaacgg ccggaagaga atgctggcct
     8281 ctgccggcga actgcagaag ggaaacgaac tggccctgcc ctccaaatat gtgaacttcc
     8341 tgtacctggc cagccactat gagaagctga agggctcccc cgaggataat gagcagaaac
     8401 agctgtttgt ggaacagcac aagcactacc tggacgagat catcgagcag atcagcgagt
     8461 tctccaagag agtgatcctg gccgacgcta atctggacaa agtgctgtcc gcctacaaca
     8521 agcaccggga taagcccatc agagagcagg ccgagaatat catccacctg tttaccctga
     8581 ccaatctggg agcccctgcc gccttcaagt actttgacac caccatcgac cggaagaggt
     8641 acaccagcac caaagaggtg ctggacgcca ccctgatcca ccagagcatc accggcctgt
     8701 acgagacacg gatcgacctg tctcagctgg gaggcgacaa gcgacctgcc gccacaaaga
     8761 aggctggaca ggctaagaag aagaaagatt acaaagacga tgacgataag ggatccggca
     8821 gcggcgccac caacttcagc ctgctgaagc aggccggcga cgtggaggag aaccccggcc
     8881 ccatggtgag caagggcgag gagctgttca ccggggtggt gcccatcctg gtcgagctgg
     8941 acggcgacgt aaacggccac aagttcagcg tgtctggcga gggcgagggc gatgccacct
     9001 acggcaagct gaccctgaag ttcatctgca ccaccggcaa gctgcccgtg ccctggccca
     9061 ccctcgtgac caccctgacc tacggcgtgc agtgcttcag ccgctacccc gaccacatga
     9121 agcagcacga cttcttcaag tccgccatgc ccgaaggcta cgtccaggag cgcaccatct
     9181 tcttcaagga cgacggcaac tacaagaccc gcgccgaggt gaagttcgag ggcgacaccc
     9241 tggtgaaccg catcgagctg aagggcatcg acttcaagga ggacggcaac atcctggggc
     9301 acaagctgga gtacaactac aacagccaca acgtctatat catggccgac aagcagaaga
     9361 acggcatcaa ggcgaacttc aagatccgcc acaacatcga ggacggcagc gtgcagctcg
     9421 ccgaccacta ccagcagaac acccccatcg gcgacggccc cgtgctgctg cccgacaacc
     9481 actacctgag cacccagtcc gccctgagca aagaccccaa cgagaaacgc gatcacatgg
     9541 tcctgctgga gttcgtgacc gccgccggga tcactctcgg catggacgag ctgtacaagt
     9601 aagaattccg caattctacc gggtagggga ggcgcttttc ccaaggcagt ctggagcatg
     9661 cgctttagca gccccgctgg gcacttggcg ctacacaagt ggcctctggc ctcgcacaca
     9721 ttccacatcc accggtaggc gccaaccggc tccgttcttt ggtggcccct tcgcgccacc
     9781 ttctactcct cccctagtca ggaagttccc ccccgccccg cagctcgcgt cgtgcaggac
     9841 gtgacaaatg gaagtagcac gtctcactag tctcgtgcag atggacagca ccgctgagca
     9901 atggaagcgg gtaggccttt ggggcagcgg ccaatagcag ctttgctcct tcgctttctg
     9961 ggctcagagg ctgggaaggg gtgggtccgg gggcgggctc aggggcgggc tcaggggcgg
    10021 ggcgggcgcc cgaaggtcct ccggaggccc ggcattctgc acgcttcaaa agcgcacgtc
    10081 tgccgcgctg ttctcctctt cctcatctcc gggcctttcg gacgtggagg agaaccccgg
    10141 ccccatgaag accttcaaca tctctcagca ggaccttgag ctggtggaag ttgccactga
    10201 gaaaatcacc atgctctatg aggacaacaa gcaccatgtt ggtgctgcca tcaggaccaa
    10261 gacaggagaa atcatctctg ctgtccacat tgaagcctac attggcaggg tcactgtgtg
    10321 tgcagaggcc attgccattg ggtctgcagt ctccaatggg cagaaggact ttgacaccat
    10381 tgtggctgtg aggcacccct acagtgatga ggtggacagg tccatcagag tggtgtcccc
    10441 ctgtggcatg tgcagggagc tcatctcaga ctatgcccct gattgctttg ttctgattga
    10501 gatgaatggc aagctggtca agacaaccat tgaggagctg attccactga aatacaccag
    10561 aaactaattc gaaagatccg ctagcgaatt cgatatcaag cttatcggta atcaacctct
    10621 ggattacaaa atttgtgaaa gattgactgg tattcttaac tatgttgctc cttttacgct
    10681 atgtggatac gctgctttaa tgcctttgta tcatgctatt gcttcccgta tggctttcat
    10741 tttctcctcc ttgtataaat cctggttgct gtctctttat gaggagttgt ggcccgttgt
    10801 caggcaacgt ggcgtggtgt gcactgtgtt tgctgacgca acccccactg gttggggcat
    10861 tgccaccacc tgtcagctcc tttccgggac tttcgctttc cccctcccta ttgccacggc
    10921 ggaactcatc gccgcctgcc ttgcccgctg ctggacaggg gctcggctgt tgggcactga
    10981 caattccgtg gtgttgtcgg ggaaatcatc gtcctttcct tggctgctcg cctgtgttgc
    11041 cacctggatt ctgcgcggga cgtccttctg ctacgtccct tcggccctca atccagcgga
    11101 ccttccttcc cgcggcctgc tgccggctct gcggcctctt ccgcgtcttc gccttcgccc
    11161 tcagacgagt cggatctccc tttgggccgc ctccccgcat cgataccgtc gacctcgaga
    11221 cctagaaaaa catggagcaa tcacaagtag caatacagca gctaccaatg ctgattgtgc
    11281 ctggctagaa gcacaagagg aggaggaggt gggttttcca gtcacacctc aggtaccttt
    11341 aagaccaatg acttacaagg cagctgtaga tcttagccac tttttaaaag aaaagggggg
    11401 actggaaggg ctaattcact cccaacgaag acaagatatc cttgatctgt ggatctacca
    11461 cacacaaggc tacttccctg attggcagaa ctacacacca gggccaggga tcagatatcc
    11521 actgaccttt ggatggtgct acaagctagt accagttgag caagagaagg tagaagaagc
    11581 caatgaagga gagaacaccc gcttgttaca ccctgtgagc ctgcatggga tggatgaccc
    11641 ggagagagaa gtattagagt ggaggtttga cagccgccta gcatttcatc acatggcccg
    11701 agagctgcat ccggactgta ctgggtctct ctggttagac cagatctgag cctgggagct
    11761 ctctggctaa ctagggaacc cactgcttaa gcctcaataa agcttgcctt gagtgcttca
    11821 agtagtgtgt gcccgtctgt tgtgtgactc tggtaactag agatccctca gaccctttta
    11881 gtcagtgtgg aaaatctcta gcagggcccg tttaaacccg ctgatcagcc tcgactgtgc
    11941 cttctagttg ccagccatct gttgtttgcc cctcccccgt gccttccttg accctggaag
    12001 gtgccactcc cactgtcctt tcctaataaa atgaggaaat tgcatcgcat tgtctgagta
    12061 ggtgtcattc tattctgggg ggtggggtgg ggcaggacag caagggggag gattgggaag
    12121 acaatagcag gcatgctggg gatgcggtgg gctctatggc ttctgaggcg gaaagaacca
    12181 gctggggctc tagggggtat ccccacgcgc cctgtagcgg cgcattaagc gcggcgggtg
    12241 tggtggttac gcgcagcgtg accgctacac ttgccagcgc cctagcgccc gctcctttcg
    12301 ctttcttccc ttcctttctc gccacgttcg ccggctttcc ccgtcaagct ctaaatcggg
    12361 ggctcccttt agggttccga tttagtgctt tacggcacct cgaccccaaa aaacttgatt
    12421 agggtgatgg ttcacgtagt gggccatcgc cctgatagac ggtttttcgc cctttgacgt
    12481 tggagtccac gttctttaat agtggactct tgttccaaac tggaacaaca ctcaacccta
    12541 tctcggtcta ttcttttgat ttataaggga ttttgccgat ttcggcctat tggttaaaaa
    12601 atgagctgat ttaacaaaaa tttaacgcga attaattctg tggaatgtgt gtcagttagg
    12661 gtgtggaaag tccccaggct ccccagcagg cagaagtatg caaagcatgc atctcaatta
    12721 gtcagcaacc aggtgtggaa agtccccagg ctccccagca ggcagaagta tgcaaagcat
    12781 gcatctcaat tagtcagcaa ccatagtccc gcccctaact ccgcccatcc cgcccctaac
    12841 tccgcccagt tccgcccatt ctccgcccca tggctgacta atttttttta tttatgcaga
    12901 ggccgaggcc gcctctgcct ctgagctatt ccagaagtag tgaggaggct tttttggagg
    12961 cctaggcttt tgcaaaaagc tcccgggagc ttgtatatcc attttcggat ctgatcagca
    13021 cgtgttgaca attaatcatc ggcatagtat atcggcatag tataatacga caaggtgagg
    13081 aactaaacca tggccaagtt gaccagtgcc gttccggtgc tcaccgcgcg cgacgtcgcc
    13141 ggagcggtcg agttctggac cgaccggctc gggttctccc gggacttcgt ggaggacgac
    13201 ttcgccggtg tggtccggga cgacgtgacc ctgttcatca gcgcggtcca ggaccaggtg
    13261 gtgccggaca acaccctggc ctgggtgtgg gtgcgcggcc tggacgagct gtacgccgag
    13321 tggtcggagg tcgtgtccac gaacttccgg gacgcctccg ggccggccat gaccgagatc
    13381 ggcgagcagc cgtgggggcg ggagttcgcc ctgcgcgacc cggccggcaa ctgcgtgcac
    13441 ttcgtggccg aggagcagga ctgacacgtg ctacgagatt tcgattccac cgccgccttc
    13501 tatgaaaggt tgggcttcgg aatcgttttc cgggacgccg gctggatgat cctccagcgc
    13561 ggggatctca tgctggagtt cttcgcccac cccaacttgt ttattgcagc ttataatggt
    13621 tacaaataaa gcaatagcat cacaaatttc acaaataaag catttttttc actgcattct
    13681 agttgtggtt tgtccaaact catcaatgta tcttatcatg tctgtatacc gtcgacctct
    13741 agctagagct tggcgtaatc atggtcatag ctgtttcctg tgtgaaattg ttatccgctc
    13801 acaattccac acaacatacg agccggaagc ataaagtgta aagcctgggg tgcctaatga
    13861 gtgagctaac tcacattaat tgcgttgcgc tcactgcccg ctttccagtc gggaaacctg
    13921 tcgtgccagc tgcattaatg aatcggccaa cgcgcgggga gaggcggttt gcgtattggg
    13981 cgctcttccg cttcctcgct cactgactcg ctgcgctcgg tcgttcggct gcggcgagcg
    14041 gtatcagctc actcaaaggc ggtaatacgg ttatccacag aatcagggga taacgcagga
    14101 aagaacatgt gagcaaaagg ccagcaaaag gccaggaacc gtaaaaaggc cgcgttgctg
    14161 gcgtttttcc ataggctccg cccccctgac gagcatcaca aaaatcgacg ctcaagtcag
    14221 aggtggcgaa acccgacagg actataaaga taccaggcgt ttccccctgg aagctccctc
    14281 gtgcgctctc ctgttccgac cctgccgctt accggatacc tgtccgcctt tctcccttcg
    14341 ggaagcgtgg cgctttctca tagctcacgc tgtaggtatc tcagttcggt gtaggtcgtt
    14401 cgctccaagc tgggctgtgt gcacgaaccc cccgttcagc ccgaccgctg cgccttatcc
    14461 ggtaactatc gtcttgagtc caacccggta agacacgact tatcgccact ggcagcagcc
    14521 actggtaaca ggattagcag agcgaggtat gtaggcggtg ctacagagtt cttgaagtgg
    14581 tggcc
//
