LOCUS       sequence-487496-       13602 bp    DNA     circular SYN 30-SEP-2025
DEFINITION  Constitutive active CRISPRgenee construct.
ACCESSION   .
VERSION     .
KEYWORDS    .
SOURCE      synthetic DNA construct
  ORGANISM  synthetic DNA construct
REFERENCE   1  (bases 1 to 13602)
  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 13602)
  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: EF1as-ZIM3-Cas9-P2A-GFP
FEATURES             Location/Qualifiers
     source          1..13602
                     /mol_type="other DNA"
                     /organism="synthetic DNA construct"
     CDS             complement(join(318..1109,1110..1178))
                     /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     941..960
                     /label=Amp-R
                     /note="Ampicillin resistance gene, reverse primer"
     promoter        complement(1179..1283)
                     /gene="bla"
                     /label=AmpR promoter
     primer_bind     complement(1358..1377)
                     /label=pRS-marker
                     /note="pRS vectors, use to sequence yeast selectable 
                     marker"
     enhancer        1549..1928
                     /label=CMV enhancer
                     /note="human cytomegalovirus immediate early enhancer"
     promoter        1930..2128
                     /label=CMV promoter
                     /note="human cytomegalovirus (CMV) immediate early 
                     promoter"
     primer_bind     2083..2103
                     /label=CMV-F
                     /note="Human CMV immediate early promoter, forward primer"
     LTR             2146..2326
                     /label=5' LTR (truncated)
                     /note="truncated 5' long terminal repeat (LTR) from HIV-1"
     misc_feature    2373..2498
                     /label=HIV-1 Psi
                     /note="packaging signal of human immunodeficiency virus 
                     type 1"
     misc_feature    2991..3224
                     /label=RRE
                     /note="The Rev response element (RRE) of HIV-1 allows for 
                     Rev-dependent mRNA export from the nucleus to the 
                     cytoplasm."
     CDS             3409..3453
                     /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    3751..3868
                     /label=cPPT/CTS
                     /note="central polypurine tract and central termination 
                     sequence of HIV-1"
     promoter        3961..4172
                     /label=EF-1-alpha core promoter
                     /note="core promoter for human elongation factor 
                     EF-1-alpha"
     protein_bind    complement(4488..4512)
                     /gene="mutant version of attB"
                     /label=attB2
                     /bound_moiety="BP Clonase(TM)"
                     /note="recombination site for the Gateway(R) BP reaction"
     CDS             4532..4552
                     /codon_start=1
                     /product="nuclear localization signal of SV40 (simian virus
                     40) large T antigen"
                     /label=SV40 NLS
                     /translation="PKKKRKV"
     regulatory      4607..4616
                     /label=Kozak sequence
                     /note="vertebrate consensus sequence for strong initiation 
                     of translation (Kozak, 1987)"
                     /regulatory_class="other"
     CDS             4613..8716
                     /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             4613..8716
                     /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             8717..8764
                     /codon_start=1
                     /product="bipartite nuclear localization signal from 
                     nucleoplasmin"
                     /label=nucleoplasmin NLS
                     /translation="KRPAATKKAGQAKKKK"
     CDS             8765..8788
                     /codon_start=1
                     /product="FLAG(R) epitope tag, followed by an enterokinase 
                     cleavage site"
                     /label=FLAG
                     /translation="DYKDDDDK"
     CDS             8804..8860
                     /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(8861..8863,8864..8866,8867..9580)
                     /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(9167..9186)
                     /label=EXFP-R
                     /note="For distinguishing EGFP variants, reverse primer"
     primer_bind     9514..9535
                     /label=EGFP-C
                     /note="EGFP, forward primer"
     misc_feature    9605..10193
                     /label=WPRE
                     /note="woodchuck hepatitis virus posttranscriptional 
                     regulatory element"
     primer_bind     complement(9658..9678)
                     /label=WPRE-R
                     /note="WPRE, reverse primer"
     CDS             complement(10076..10087)
                     /codon_start=1
                     /product="Factor Xa recognition and cleavage site"
                     /label=Factor Xa site
                     /translation="IEGR"
     primer_bind     complement(10196..10212)
                     /label=KS primer
                     /note="common sequencing primer, one of multiple similar 
                     variants"
     primer_bind     complement(10197..10213)
                     /label=pBluescriptKS
                     /note="For pBluescript vector"
     LTR             10718..10898
                     /label=5' LTR (truncated)
                     /note="truncated 5' long terminal repeat (LTR) from HIV-1"
     primer_bind     complement(10924..10941)
                     /label=BGH-rev
                     /note="Bovine growth hormone terminator, reverse primer. 
                     Also called BGH reverse"
     polyA_signal    10930..11154
                     /label=bGH poly(A) signal
                     /note="bovine growth hormone polyadenylation signal"
     rep_origin      11200..11628
                     /direction=RIGHT
                     /label=f1 ori
                     /note="f1 bacteriophage origin of replication; arrow 
                     indicates direction of (+) strand synthesis"
     primer_bind     complement(11287..11306)
                     /label=F1ori-R
                     /note="F1 origin, reverse primer"
     primer_bind     11497..11518
                     /label=F1ori-F
                     /note="F1 origin, forward primer"
     primer_bind     complement(11637..11657)
                     /label=pBABE 3'
                     /note="SV40 enhancer, reverse primer for pBABE vectors"
     promoter        11642..11971
                     /label=SV40 promoter
                     /note="SV40 enhancer and early promoter"
     rep_origin      11822..11957
                     /label=SV40 ori
                     /note="SV40 origin of replication"
     primer_bind     11884..11903
                     /label=SV40pro-F
                     /note="SV40 promoter/origin, forward primer"
     promoter        12019..12066
                     /label=EM7 promoter
                     /note="synthetic bacterial promoter "
     CDS             12085..12459
                     /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    12589..12710
                     /label=SV40 poly(A) signal
                     /note="SV40 polyadenylation signal"
     primer_bind     complement(12626..12645)
                     /label=SV40pA-R
                     /note="SV40 polyA, reverse primer"
     primer_bind     12680..12699
                     /label=EBV-rev
                     /note="SV40 polyA terminator, reverse primer"
     primer_bind     complement(12759..12775)
                     /label=M13 rev
                     /note="common sequencing primer, one of multiple similar 
                     variants"
     primer_bind     complement(12759..12775)
                     /label=M13 Reverse
                     /note="In lacZ gene. Also called M13-rev"
     primer_bind     complement(12772..12794)
                     /label=M13/pUC Reverse
                     /note="In lacZ gene"
     protein_bind    12783..12799
                     /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(12807..12813,12814..12831,12832..12837))
                     /label=lac promoter
                     /note="promoter for the E. coli lac operon"
     protein_bind    12852..12873
                     /label=CAP binding site
                     /bound_moiety="E. coli catabolite activator protein"
                     /note="CAP binding activates transcription in the presence 
                     of cAMP."
     primer_bind     complement(12990..13007)
                     /label=L4440
                     /note="L4440 vector, forward primer"
     rep_origin      complement(join(13161..13602,1..147))
                     /direction=LEFT
                     /label=ori
                     /note="high-copy-number ColE1/pMB1/pBR322/pUC origin of 
                     replication"
     primer_bind     complement(13241..13260)
                     /label=pBR322ori-F
                     /note="pBR322 origin, forward primer"
ORIGIN
        1 acagtatttg gtatctgcgc tctgctgaag ccagttacct tcggaaaaag agttggtagc
       61 tcttgatccg gcaaacaaac caccgctggt agcggtggtt tttttgtttg caagcagcag
      121 attacgcgca gaaaaaaagg atctcaagaa gatcctttga tcttttctac ggggtctgac
      181 gctcagtgga acgaaaactc acgttaaggg attttggtca tgagattatc aaaaaggatc
      241 ttcacctaga tccttttaaa ttaaaaatga agttttaaat caatctaaag tatatatgag
      301 taaacttggt ctgacagtta ccaatgctta atcagtgagg cacctatctc agcgatctgt
      361 ctatttcgtt catccatagt tgcctgactc cccgtcgtgt agataactac gatacgggag
      421 ggcttaccat ctggccccag tgctgcaatg ataccgcgag acccacgctc accggctcca
      481 gatttatcag caataaacca gccagccgga agggccgagc gcagaagtgg tcctgcaact
      541 ttatccgcct ccatccagtc tattaattgt tgccgggaag ctagagtaag tagttcgcca
      601 gttaatagtt tgcgcaacgt tgttgccatt gctacaggca tcgtggtgtc acgctcgtcg
      661 tttggtatgg cttcattcag ctccggttcc caacgatcaa ggcgagttac atgatccccc
      721 atgttgtgca aaaaagcggt tagctccttc ggtcctccga tcgttgtcag aagtaagttg
      781 gccgcagtgt tatcactcat ggttatggca gcactgcata attctcttac tgtcatgcca
      841 tccgtaagat gcttttctgt gactggtgag tactcaacca agtcattctg agaatagtgt
      901 atgcggcgac cgagttgctc ttgcccggcg tcaatacggg ataataccgc gccacatagc
      961 agaactttaa aagtgctcat cattggaaaa cgttcttcgg ggcgaaaact ctcaaggatc
     1021 ttaccgctgt tgagatccag ttcgatgtaa cccactcgtg cacccaactg atcttcagca
     1081 tcttttactt tcaccagcgt ttctgggtga gcaaaaacag gaaggcaaaa tgccgcaaaa
     1141 aagggaataa gggcgacacg gaaatgttga atactcatac tcttcctttt tcaatattat
     1201 tgaagcattt atcagggtta ttgtctcatg agcggataca tatttgaatg tatttagaaa
     1261 aataaacaaa taggggttcc gcgcacattt ccccgaaaag tgccacctga cgtcgacgga
     1321 tcgggagatc tcccgatccc ctatggtgca ctctcagtac aatctgctct gatgccgcat
     1381 agttaagcca gtatctgctc cctgcttgtg tgttggaggt cgctgagtag tgcgcgagca
     1441 aaatttaagc tacaacaagg caaggcttga ccgacaattg catgaagaat ctgcttaggg
     1501 ttaggcgttt tgcgctgctt cgcgatgtac gggccagata tacgcgttga cattgattat
     1561 tgactagtta ttaatagtaa tcaattacgg ggtcattagt tcatagccca tatatggagt
     1621 tccgcgttac ataacttacg gtaaatggcc cgcctggctg accgcccaac gacccccgcc
     1681 cattgacgtc aataatgacg tatgttccca tagtaacgcc aatagggact ttccattgac
     1741 gtcaatgggt ggagtattta cggtaaactg cccacttggc agtacatcaa gtgtatcata
     1801 tgccaagtac gccccctatt gacgtcaatg acggtaaatg gcccgcctgg cattatgccc
     1861 agtacatgac cttatgggac tttcctactt ggcagtacat ctacgtatta gtcatcgcta
     1921 ttaccatggt gatgcggttt tggcagtaca tcaatgggcg tggatagcgg tttgactcac
     1981 ggggatttcc aagtctccac cccattgacg tcaatgggag tttgttttgg caccaaaatc
     2041 aacgggactt tccaaaatgt cgtaacaact ccgccccatt gacgcaaatg ggcggtaggc
     2101 gtgtacggtg ggaggtctat ataagcagcg cgttttgcct gtactgggtc tctctggtta
     2161 gaccagatct gagcctggga gctctctggc taactaggga acccactgct taagcctcaa
     2221 taaagcttgc cttgagtgct tcaagtagtg tgtgcccgtc tgttgtgtga ctctggtaac
     2281 tagagatccc tcagaccctt ttagtcagtg tggaaaatct ctagcagtgg cgcccgaaca
     2341 gggacttgaa agcgaaaggg aaaccagagg agctctctcg acgcaggact cggcttgctg
     2401 aagcgcgcac ggcaagaggc gaggggcggc gactggtgag tacgccaaaa attttgacta
     2461 gcggaggcta gaaggagaga gatgggtgcg agagcgtcag tattaagcgg gggagaatta
     2521 gatcgcgatg ggaaaaaatt cggttaaggc cagggggaaa gaaaaaatat aaattaaaac
     2581 atatagtatg ggcaagcagg gagctagaac gattcgcagt taatcctggc ctgttagaaa
     2641 catcagaagg ctgtagacaa atactgggac agctacaacc atcccttcag acaggatcag
     2701 aagaacttag atcattatat aatacagtag caaccctcta ttgtgtgcat caaaggatag
     2761 agataaaaga caccaaggaa gctttagaca agatagagga agagcaaaac aaaagtaaga
     2821 ccaccgcaca gcaagcggcc gctgatcttc agacctggag gaggagatat gagggacaat
     2881 tggagaagtg aattatataa atataaagta gtaaaaattg aaccattagg agtagcaccc
     2941 accaaggcaa agagaagagt ggtgcagaga gaaaaaagag cagtgggaat aggagctttg
     3001 ttccttgggt tcttgggagc agcaggaagc actatgggcg cagcgtcaat gacgctgacg
     3061 gtacaggcca gacaattatt gtctggtata gtgcagcagc agaacaattt gctgagggct
     3121 attgaggcgc aacagcatct gttgcaactc acagtctggg gcatcaagca gctccaggca
     3181 agaatcctgg ctgtggaaag atacctaaag gatcaacagc tcctggggat ttggggttgc
     3241 tctggaaaac tcatttgcac cactgctgtg ccttggaatg ctagttggag taataaatct
     3301 ctggaacaga tttggaatca cacgacctgg atggagtggg acagagaaat taacaattac
     3361 acaagcttaa tacactcctt aattgaagaa tcgcaaaacc agcaagaaaa gaatgaacaa
     3421 gaattattgg aattagataa atgggcaagt ttgtggaatt ggtttaacat aacaaattgg
     3481 ctgtggtata taaaattatt cataatgata gtaggaggct tggtaggttt aagaatagtt
     3541 tttgctgtac tttctatagt gaatagagtt aggcagggat attcaccatt atcgtttcag
     3601 acccacctcc caaccccgag gggacccgac aggcccgaag gaatagaaga agaaggtgga
     3661 gagagagaca gagacagatc cattcgatta gtgaacggat cggcactgcg tgcgccaatt
     3721 ctgcagacaa atggcagtat tcatccacaa ttttaaaaga aaagggggga ttggggggta
     3781 cagtgcaggg gaaagaatag tagacataat agcaacagac atacaaacta aagaattaca
     3841 aaaacaaatt acaaaaattc aaaattttcg ggtttattac agggacagca gagatccagt
     3901 ttggttaatt agctagctag gtcttgaaag gagtgggaat tggctccggt gcccgtcagt
     3961 gggcagagcg cacatcgccc acagtccccg agaagttggg gggaggggtc ggcaattgat
     4021 ccggtgccta gagaaggtgg cgcggggtaa actgggaaag tgatgtcgtg tactggctcc
     4081 gcctttttcc cgagggtggg ggagaaccgt atataagtgc agtagtcgcc gtgaacgttc
     4141 tttttcgcaa cgggtttgcc gccagaacac aggaccggtt gccaccatga acaattccca
     4201 gggaagagtg accttcgagg atgtcactgt gaacttcacc cagggggagt ggcagcggct
     4261 gaatcccgaa cagagaaact tgtacaggga tgtgatgctg gagaattaca gcaaccttgt
     4321 ctctgtggga caaggggaaa ccaccaaacc cgatgtgatc ttgaggttgg aacaaggaaa
     4381 ggagccatgg ttggaggaag aggaagtgct gggaagtggc cgtgcagaaa aaaatgggga
     4441 cattggaggg cagatttgga agccaaagga tgtgaaagag agtctctacc caactttctt
     4501 gtacaaagtg gttggtggat ctggaggttc tcccaagaag aagcgcaagg tgggacgcgt
     4561 ctgcaggata tcaagcttgc ggtaccgcgg gcccgggatc agcgctgcca ccatggacaa
     4621 gaagtacagc atcggcctgg acatcggcac caactctgtg ggctgggccg tgatcaccga
     4681 cgagtacaag gtgcccagca agaaattcaa ggtgctgggc aacaccgacc ggcacagcat
     4741 caagaagaac ctgatcggag ccctgctgtt cgacagcggc gaaacagccg aggccacccg
     4801 gctgaagaga accgccagaa gaagatacac cagacggaag aaccggatct gctatctgca
     4861 agagatcttc agcaacgaga tggccaaggt ggacgacagc ttcttccaca gactggaaga
     4921 gtccttcctg gtggaagagg ataagaagca cgagcggcac cccatcttcg gcaacatcgt
     4981 ggacgaggtg gcctaccacg agaagtaccc caccatctac cacctgagaa agaaactggt
     5041 ggacagcacc gacaaggccg acctgcggct gatctatctg gccctggccc acatgatcaa
     5101 gttccggggc cacttcctga tcgagggcga cctgaacccc gacaacagcg acgtggacaa
     5161 gctgttcatc cagctggtgc agacctacaa ccagctgttc gaggaaaacc ccatcaacgc
     5221 cagcggcgtg gacgccaagg ccatcctgtc tgccagactg agcaagagca gacggctgga
     5281 aaatctgatc gcccagctgc ccggcgagaa gaagaatggc ctgttcggaa acctgattgc
     5341 cctgagcctg ggcctgaccc ccaacttcaa gagcaacttc gacctggccg aggatgccaa
     5401 actgcagctg agcaaggaca cctacgacga cgacctggac aacctgctgg cccagatcgg
     5461 cgaccagtac gccgacctgt ttctggccgc caagaacctg tccgacgcca tcctgctgag
     5521 cgacatcctg agagtgaaca ccgagatcac caaggccccc ctgagcgcct ctatgatcaa
     5581 gagatacgac gagcaccacc aggacctgac cctgctgaaa gctctcgtgc ggcagcagct
     5641 gcctgagaag tacaaagaga ttttcttcga ccagagcaag aacggctacg ccggctacat
     5701 tgacggcgga gccagccagg aagagttcta caagttcatc aagcccatcc tggaaaagat
     5761 ggacggcacc gaggaactgc tcgtgaagct gaacagagag gacctgctgc ggaagcagcg
     5821 gaccttcgac aacggcagca tcccccacca gatccacctg ggagagctgc acgccattct
     5881 gcggcggcag gaagattttt acccattcct gaaggacaac cgggaaaaga tcgagaagat
     5941 cctgaccttc cgcatcccct actacgtggg ccctctggcc aggggaaaca gcagattcgc
     6001 ctggatgacc agaaagagcg aggaaaccat caccccctgg aacttcgagg aagtggtgga
     6061 caagggcgct tccgcccaga gcttcatcga gcggatgacc aacttcgata agaacctgcc
     6121 caacgagaag gtgctgccca agcacagcct gctgtacgag tacttcaccg tgtataacga
     6181 gctgaccaaa gtgaaatacg tgaccgaggg aatgagaaag cccgccttcc tgagcggcga
     6241 gcagaaaaag gccatcgtgg acctgctgtt caagaccaac cggaaagtga ccgtgaagca
     6301 gctgaaagag gactacttca agaaaatcga gtgcttcgac tccgtggaaa tctccggcgt
     6361 ggaagatcgg ttcaacgcct ccctgggcac ataccacgat ctgctgaaaa ttatcaagga
     6421 caaggacttc ctggacaatg aggaaaacga ggacattctg gaagatatcg tgctgaccct
     6481 gacactgttt gaggacagag agatgatcga ggaacggctg aaaacctatg cccacctgtt
     6541 cgacgacaaa gtgatgaagc agctgaagcg gcggagatac accggctggg gcaggctgag
     6601 ccggaagctg atcaacggca tccgggacaa gcagtccggc aagacaatcc tggatttcct
     6661 gaagtccgac ggcttcgcca acagaaactt catgcagctg atccacgacg acagcctgac
     6721 ctttaaagag gacatccaga aagcccaggt gtccggccag ggcgatagcc tgcacgagca
     6781 cattgccaat ctggccggca gccccgccat taagaagggc atcctgcaga cagtgaaggt
     6841 ggtggacgag ctcgtgaaag tgatgggccg gcacaagccc gagaacatcg tgatcgaaat
     6901 ggccagagag aaccagacca cccagaaggg acagaagaac agccgcgaga gaatgaagcg
     6961 gatcgaagag ggcatcaaag agctgggcag ccagatcctg aaagaacacc ccgtggaaaa
     7021 cacccagctg cagaacgaga agctgtacct gtactacctg cagaatgggc gggatatgta
     7081 cgtggaccag gaactggaca tcaaccggct gtccgactac gatgtggacc atatcgtgcc
     7141 tcagagcttt ctgaaggacg actccatcga caacaaggtg ctgaccagaa gcgacaagaa
     7201 ccggggcaag agcgacaacg tgccctccga agaggtcgtg aagaagatga agaactactg
     7261 gcggcagctg ctgaacgcca agctgattac ccagagaaag ttcgacaatc tgaccaaggc
     7321 cgagagaggc ggcctgagcg aactggataa ggccggcttc atcaagagac agctggtgga
     7381 aacccggcag atcacaaagc acgtggcaca gatcctggac tcccggatga acactaagta
     7441 cgacgagaat gacaagctga tccgggaagt gaaagtgatc accctgaagt ccaagctggt
     7501 gtccgatttc cggaaggatt tccagtttta caaagtgcgc gagatcaaca actaccacca
     7561 cgcccacgac gcctacctga acgccgtcgt gggaaccgcc ctgatcaaaa agtaccctaa
     7621 gctggaaagc gagttcgtgt acggcgacta caaggtgtac gacgtgcgga agatgatcgc
     7681 caagagcgag caggaaatcg gcaaggctac cgccaagtac ttcttctaca gcaacatcat
     7741 gaactttttc aagaccgaga ttaccctggc caacggcgag atccggaagc ggcctctgat
     7801 cgagacaaac ggcgaaaccg gggagatcgt gtgggataag ggccgggatt ttgccaccgt
     7861 gcggaaagtg ctgagcatgc cccaagtgaa tatcgtgaaa aagaccgagg tgcagacagg
     7921 cggcttcagc aaagagtcta tcctgcccaa gaggaacagc gataagctga tcgccagaaa
     7981 gaaggactgg gaccctaaga agtacggcgg cttcgacagc cccaccgtgg cctattctgt
     8041 gctggtggtg gccaaagtgg aaaagggcaa gtccaagaaa ctgaagagtg tgaaagagct
     8101 actggggatc accatcatgg aaagaagcag cttcgagaag aatcccatcg actttctgga
     8161 agccaagggc tacaaagaag tgaaaaagga cctgatcatc aagctgccta agtactccct
     8221 gttcgagctg gaaaacggcc ggaagagaat gctggcctct gccggcgaac tgcagaaggg
     8281 aaacgaactg gccctgccct ccaaatatgt gaacttcctg tacctggcca gccactatga
     8341 gaagctgaag ggctcccccg aggataatga gcagaaacag ctgtttgtgg aacagcacaa
     8401 gcactacctg gacgagatca tcgagcagat cagcgagttc tccaagagag tgatcctggc
     8461 cgacgctaat ctggacaaag tgctgtccgc ctacaacaag caccgggata agcccatcag
     8521 agagcaggcc gagaatatca tccacctgtt taccctgacc aatctgggag cccctgccgc
     8581 cttcaagtac tttgacacca ccatcgaccg gaagaggtac accagcacca aagaggtgct
     8641 ggacgccacc ctgatccacc agagcatcac cggcctgtac gagacacgga tcgacctgtc
     8701 tcagctggga ggcgacaagc gacctgccgc cacaaagaag gctggacagg ctaagaagaa
     8761 gaaagattac aaagacgatg acgataaggg atccggcagc ggcgccacca acttcagcct
     8821 gctgaagcag gccggcgacg tggaggagaa ccccggcccc atggtgagca agggcgagga
     8881 gctgttcacc ggggtggtgc ccatcctggt cgagctggac ggcgacgtaa acggccacaa
     8941 gttcagcgtg tctggcgagg gcgagggcga tgccacctac ggcaagctga ccctgaagtt
     9001 catctgcacc accggcaagc tgcccgtgcc ctggcccacc ctcgtgacca ccctgaccta
     9061 cggcgtgcag tgcttcagcc gctaccccga ccacatgaag cagcacgact tcttcaagtc
     9121 cgccatgccc gaaggctacg tccaggagcg caccatcttc ttcaaggacg acggcaacta
     9181 caagacccgc gccgaggtga agttcgaggg cgacaccctg gtgaaccgca tcgagctgaa
     9241 gggcatcgac ttcaaggagg acggcaacat cctggggcac aagctggagt acaactacaa
     9301 cagccacaac gtctatatca tggccgacaa gcagaagaac ggcatcaagg cgaacttcaa
     9361 gatccgccac aacatcgagg acggcagcgt gcagctcgcc gaccactacc agcagaacac
     9421 ccccatcggc gacggccccg tgctgctgcc cgacaaccac tacctgagca cccagtccgc
     9481 cctgagcaaa gaccccaacg agaaacgcga tcacatggtc ctgctggagt tcgtgaccgc
     9541 cgccgggatc actctcggca tggacgagct gtacaagtaa gaattcgata tcaagcttat
     9601 cggtaatcaa cctctggatt acaaaatttg tgaaagattg actggtattc ttaactatgt
     9661 tgctcctttt acgctatgtg gatacgctgc tttaatgcct ttgtatcatg ctattgcttc
     9721 ccgtatggct ttcattttct cctccttgta taaatcctgg ttgctgtctc tttatgagga
     9781 gttgtggccc gttgtcaggc aacgtggcgt ggtgtgcact gtgtttgctg acgcaacccc
     9841 cactggttgg ggcattgcca ccacctgtca gctcctttcc gggactttcg ctttccccct
     9901 ccctattgcc acggcggaac tcatcgccgc ctgccttgcc cgctgctgga caggggctcg
     9961 gctgttgggc actgacaatt ccgtggtgtt gtcggggaaa tcatcgtcct ttccttggct
    10021 gctcgcctgt gttgccacct ggattctgcg cgggacgtcc ttctgctacg tcccttcggc
    10081 cctcaatcca gcggaccttc cttcccgcgg cctgctgccg gctctgcggc ctcttccgcg
    10141 tcttcgcctt cgccctcaga cgagtcggat ctccctttgg gccgcctccc cgcatcgata
    10201 ccgtcgacct cgagacctag aaaaacatgg agcaatcaca agtagcaata cagcagctac
    10261 caatgctgat tgtgcctggc tagaagcaca agaggaggag gaggtgggtt ttccagtcac
    10321 acctcaggta cctttaagac caatgactta caaggcagct gtagatctta gccacttttt
    10381 aaaagaaaag gggggactgg aagggctaat tcactcccaa cgaagacaag atatccttga
    10441 tctgtggatc taccacacac aaggctactt ccctgattgg cagaactaca caccagggcc
    10501 agggatcaga tatccactga cctttggatg gtgctacaag ctagtaccag ttgagcaaga
    10561 gaaggtagaa gaagccaatg aaggagagaa cacccgcttg ttacaccctg tgagcctgca
    10621 tgggatggat gacccggaga gagaagtatt agagtggagg tttgacagcc gcctagcatt
    10681 tcatcacatg gcccgagagc tgcatccgga ctgtactggg tctctctggt tagaccagat
    10741 ctgagcctgg gagctctctg gctaactagg gaacccactg cttaagcctc aataaagctt
    10801 gccttgagtg cttcaagtag tgtgtgcccg tctgttgtgt gactctggta actagagatc
    10861 cctcagaccc ttttagtcag tgtggaaaat ctctagcagg gcccgtttaa acccgctgat
    10921 cagcctcgac tgtgccttct agttgccagc catctgttgt ttgcccctcc cccgtgcctt
    10981 ccttgaccct ggaaggtgcc actcccactg tcctttccta ataaaatgag gaaattgcat
    11041 cgcattgtct gagtaggtgt cattctattc tggggggtgg ggtggggcag gacagcaagg
    11101 gggaggattg ggaagacaat agcaggcatg ctggggatgc ggtgggctct atggcttctg
    11161 aggcggaaag aaccagctgg ggctctaggg ggtatcccca cgcgccctgt agcggcgcat
    11221 taagcgcggc gggtgtggtg gttacgcgca gcgtgaccgc tacacttgcc agcgccctag
    11281 cgcccgctcc tttcgctttc ttcccttcct ttctcgccac gttcgccggc tttccccgtc
    11341 aagctctaaa tcgggggctc cctttagggt tccgatttag tgctttacgg cacctcgacc
    11401 ccaaaaaact tgattagggt gatggttcac gtagtgggcc atcgccctga tagacggttt
    11461 ttcgcccttt gacgttggag tccacgttct ttaatagtgg actcttgttc caaactggaa
    11521 caacactcaa ccctatctcg gtctattctt ttgatttata agggattttg ccgatttcgg
    11581 cctattggtt aaaaaatgag ctgatttaac aaaaatttaa cgcgaattaa ttctgtggaa
    11641 tgtgtgtcag ttagggtgtg gaaagtcccc aggctcccca gcaggcagaa gtatgcaaag
    11701 catgcatctc aattagtcag caaccaggtg tggaaagtcc ccaggctccc cagcaggcag
    11761 aagtatgcaa agcatgcatc tcaattagtc agcaaccata gtcccgcccc taactccgcc
    11821 catcccgccc ctaactccgc ccagttccgc ccattctccg ccccatggct gactaatttt
    11881 ttttatttat gcagaggccg aggccgcctc tgcctctgag ctattccaga agtagtgagg
    11941 aggctttttt ggaggcctag gcttttgcaa aaagctcccg ggagcttgta tatccatttt
    12001 cggatctgat cagcacgtgt tgacaattaa tcatcggcat agtatatcgg catagtataa
    12061 tacgacaagg tgaggaacta aaccatggcc aagttgacca gtgccgttcc ggtgctcacc
    12121 gcgcgcgacg tcgccggagc ggtcgagttc tggaccgacc ggctcgggtt ctcccgggac
    12181 ttcgtggagg acgacttcgc cggtgtggtc cgggacgacg tgaccctgtt catcagcgcg
    12241 gtccaggacc aggtggtgcc ggacaacacc ctggcctggg tgtgggtgcg cggcctggac
    12301 gagctgtacg ccgagtggtc ggaggtcgtg tccacgaact tccgggacgc ctccgggccg
    12361 gccatgaccg agatcggcga gcagccgtgg gggcgggagt tcgccctgcg cgacccggcc
    12421 ggcaactgcg tgcacttcgt ggccgaggag caggactgac acgtgctacg agatttcgat
    12481 tccaccgccg ccttctatga aaggttgggc ttcggaatcg ttttccggga cgccggctgg
    12541 atgatcctcc agcgcgggga tctcatgctg gagttcttcg cccaccccaa cttgtttatt
    12601 gcagcttata atggttacaa ataaagcaat agcatcacaa atttcacaaa taaagcattt
    12661 ttttcactgc attctagttg tggtttgtcc aaactcatca atgtatctta tcatgtctgt
    12721 ataccgtcga cctctagcta gagcttggcg taatcatggt catagctgtt tcctgtgtga
    12781 aattgttatc cgctcacaat tccacacaac atacgagccg gaagcataaa gtgtaaagcc
    12841 tggggtgcct aatgagtgag ctaactcaca ttaattgcgt tgcgctcact gcccgctttc
    12901 cagtcgggaa acctgtcgtg ccagctgcat taatgaatcg gccaacgcgc ggggagaggc
    12961 ggtttgcgta ttgggcgctc ttccgcttcc tcgctcactg actcgctgcg ctcggtcgtt
    13021 cggctgcggc gagcggtatc agctcactca aaggcggtaa tacggttatc cacagaatca
    13081 ggggataacg caggaaagaa catgtgagca aaaggccagc aaaaggccag gaaccgtaaa
    13141 aaggccgcgt tgctggcgtt tttccatagg ctccgccccc ctgacgagca tcacaaaaat
    13201 cgacgctcaa gtcagaggtg gcgaaacccg acaggactat aaagatacca ggcgtttccc
    13261 cctggaagct ccctcgtgcg ctctcctgtt ccgaccctgc cgcttaccgg atacctgtcc
    13321 gcctttctcc cttcgggaag cgtggcgctt tctcatagct cacgctgtag gtatctcagt
    13381 tcggtgtagg tcgttcgctc caagctgggc tgtgtgcacg aaccccccgt tcagcccgac
    13441 cgctgcgcct tatccggtaa ctatcgtctt gagtccaacc cggtaagaca cgacttatcg
    13501 ccactggcag cagccactgg taacaggatt agcagagcga ggtatgtagg cggtgctaca
    13561 gagttcttga agtggtggcc taactacggc tacactagaa ga
//
