LOCUS       sequence-207987-       13704 bp    DNA     circular SYN 15-DEC-2025
DEFINITION  single-vector CROPseq system with high fidelity SpCas9 (HypaSpCas9) 
            for use in the CROPseq CRISPR knockout screen.
ACCESSION   .
VERSION     .
KEYWORDS    .
SOURCE      synthetic DNA construct
  ORGANISM  synthetic DNA construct
REFERENCE   1  (bases 1 to 13704)
  TITLE     CROPseq plasmids
  JOURNAL   Unpublished
REFERENCE   2  (bases 1 to 13704)
  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: 
            "Unpublished"
COMMENT     Sequence Label: CROPseq-EFS-HypaSpCas9-P2A-EGFP
FEATURES             Location/Qualifiers
     source          1..13704
                     /mol_type="other DNA"
                     /organism="synthetic DNA construct"
     promoter        1..227
                     /label=RSV promoter
                     /note="Rous sarcoma virus enhancer/promoter"
     LTR             228..408
                     /label=5' LTR (truncated)
                     /note="truncated 5' long terminal repeat (LTR) from HIV-1"
     misc_feature    455..580
                     /label=HIV-1 Psi
                     /note="packaging signal of human immunodeficiency virus 
                     type 1"
     misc_feature    1073..1306
                     /label=RRE
                     /note="The Rev response element (RRE) of HIV-1 allows for 
                     Rev-dependent mRNA export from the nucleus to the 
                     cytoplasm."
     CDS             1491..1535
                     /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    1716..1833
                     /label=cPPT/CTS
                     /note="central polypurine tract and central termination 
                     sequence of HIV-1"
     promoter        2022..2233
                     /label=EF-1-alpha core promoter
                     /note="core promoter for human elongation factor 
                     EF-1-alpha"
     regulatory      2252..2261
                     /label=Kozak sequence
                     /note="vertebrate consensus sequence for strong initiation 
                     of translation (Kozak, 1987)"
                     /regulatory_class="other"
     CDS             join(2258..4330,4331..4333,4334..4336,4337..4342,4343..4348,
                     4349..4351,4352..6361)
                     /codon_start=1
                     /product="Cas9 endonuclease from the Streptococcus pyogenes
                     Type II CRISPR/Cas system, mutated to improve targeting 
                     specificity without compromising on-target activity (Chen 
                     et al., 2017)"
                     /label=HypaCas9
                     /note="carries the mutations N692A, M694A, Q695A, and 
                     H698A"
                     /translation="MDKKYSIGLDIGTNSVGWAVITDEYKVPSKKFKVLGNTDRHSIKK
                     NLIGALLFDSGETAEATRLKRTARRRYTRRKNRICYLQEIFSNEMAKVDDSFFHRLEES
                     FLVEEDKKHERHPIFGNIVDEVAYHEKYPTIYHLRKKLVDSTDKADLRLIYLALAHMIK
                     FRGHFLIEGDLNPDNSDVDKLFIQLVQTYNQLFEENPINASGVDAKAILSARLSKSRRL
                     ENLIAQLPGEKKNGLFGNLIALSLGLTPNFKSNFDLAEDAKLQLSKDTYDDDLDNLLAQ
                     IGDQYADLFLAAKNLSDAILLSDILRVNTEITKAPLSASMIKRYDEHHQDLTLLKALVR
                     QQLPEKYKEIFFDQSKNGYAGYIDGGASQEEFYKFIKPILEKMDGTEELLVKLNREDLL
                     RKQRTFDNGSIPHQIHLGELHAILRRQEDFYPFLKDNREKIEKILTFRIPYYVGPLARG
                     NSRFAWMTRKSEETITPWNFEEVVDKGASAQSFIERMTNFDKNLPNEKVLPKHSLLYEY
                     FTVYNELTKVKYVTEGMRKPAFLSGEQKKAIVDLLFKTNRKVTVKQLKEDYFKKIECFD
                     SVEISGVEDRFNASLGTYHDLLKIIKDKDFLDNEENEDILEDIVLTLTLFEDREMIEER
                     LKTYAHLFDDKVMKQLKRRRYTGWGRLSRKLINGIRDKQSGKTILDFLKSDGFANRAFA
                     ALIADDSLTFKEDIQKAQVSGQGDSLHEHIANLAGSPAIKKGILQTVKVVDELVKVMGR
                     HKPENIVIEMARENQTTQKGQKNSRERMKRIEEGIKELGSQILKEHPVENTQLQNEKLY
                     LYYLQNGRDMYVDQELDINRLSDYDVDHIVPQSFLKDDSIDNKVLTRSDKNRGKSDNVP
                     SEEVVKKMKNYWRQLLNAKLITQRKFDNLTKAERGGLSELDKAGFIKRQLVETRQITKH
                     VAQILDSRMNTKYDENDKLIREVKVITLKSKLVSDFRKDFQFYKVREINNYHHAHDAYL
                     NAVVGTALIKKYPKLESEFVYGDYKVYDVRKMIAKSEQEIGKATAKYFFYSNIMNFFKT
                     EITLANGEIRKRPLIETNGETGEIVWDKGRDFATVRKVLSMPQVNIVKKTEVQTGGFSK
                     ESILPKRNSDKLIARKKDWDPKKYGGFDSPTVAYSVLVVAKVEKGKSKKLKSVKELLGI
                     TIMERSSFEKNPIDFLEAKGYKEVKKDLIIKLPKYSLFELENGRKRMLASAGELQKGNE
                     LALPSKYVNFLYLASHYEKLKGSPEDNEQKQLFVEQHKHYLDEIIEQISEFSKRVILAD
                     ANLDKVLSAYNKHRDKPIREQAENIIHLFTLTNLGAPAAFKYFDTTIDRKRYTSTKEVL
                     DATLIHQSITGLYETRIDLSQLGGD"
     CDS             join(2258..3745,3746..3748,3749..4237,4238..4240,4241..4339,
                     4340..4342,4343..5032,5033..5035,5036..6361)
                     /codon_start=1
                     /product="Cas9 endonuclease from the Streptococcus pyogenes
                     Type II CRISPR/Cas system, mutated to improve targeting 
                     specificity (Kleinstiver et al., 2016)"
                     /label=SpCas9-HF1
                     /note="carries the mutations N497A, R661A, Q695A, and 
                     Q926A"
                     /translation="MDKKYSIGLDIGTNSVGWAVITDEYKVPSKKFKVLGNTDRHSIKK
                     NLIGALLFDSGETAEATRLKRTARRRYTRRKNRICYLQEIFSNEMAKVDDSFFHRLEES
                     FLVEEDKKHERHPIFGNIVDEVAYHEKYPTIYHLRKKLVDSTDKADLRLIYLALAHMIK
                     FRGHFLIEGDLNPDNSDVDKLFIQLVQTYNQLFEENPINASGVDAKAILSARLSKSRRL
                     ENLIAQLPGEKKNGLFGNLIALSLGLTPNFKSNFDLAEDAKLQLSKDTYDDDLDNLLAQ
                     IGDQYADLFLAAKNLSDAILLSDILRVNTEITKAPLSASMIKRYDEHHQDLTLLKALVR
                     QQLPEKYKEIFFDQSKNGYAGYIDGGASQEEFYKFIKPILEKMDGTEELLVKLNREDLL
                     RKQRTFDNGSIPHQIHLGELHAILRRQEDFYPFLKDNREKIEKILTFRIPYYVGPLARG
                     NSRFAWMTRKSEETITPWNFEEVVDKGASAQSFIERMTNFDKNLPNEKVLPKHSLLYEY
                     FTVYNELTKVKYVTEGMRKPAFLSGEQKKAIVDLLFKTNRKVTVKQLKEDYFKKIECFD
                     SVEISGVEDRFNASLGTYHDLLKIIKDKDFLDNEENEDILEDIVLTLTLFEDREMIEER
                     LKTYAHLFDDKVMKQLKRRRYTGWGRLSRKLINGIRDKQSGKTILDFLKSDGFANRAFA
                     ALIADDSLTFKEDIQKAQVSGQGDSLHEHIANLAGSPAIKKGILQTVKVVDELVKVMGR
                     HKPENIVIEMARENQTTQKGQKNSRERMKRIEEGIKELGSQILKEHPVENTQLQNEKLY
                     LYYLQNGRDMYVDQELDINRLSDYDVDHIVPQSFLKDDSIDNKVLTRSDKNRGKSDNVP
                     SEEVVKKMKNYWRQLLNAKLITQRKFDNLTKAERGGLSELDKAGFIKRQLVETRQITKH
                     VAQILDSRMNTKYDENDKLIREVKVITLKSKLVSDFRKDFQFYKVREINNYHHAHDAYL
                     NAVVGTALIKKYPKLESEFVYGDYKVYDVRKMIAKSEQEIGKATAKYFFYSNIMNFFKT
                     EITLANGEIRKRPLIETNGETGEIVWDKGRDFATVRKVLSMPQVNIVKKTEVQTGGFSK
                     ESILPKRNSDKLIARKKDWDPKKYGGFDSPTVAYSVLVVAKVEKGKSKKLKSVKELLGI
                     TIMERSSFEKNPIDFLEAKGYKEVKKDLIIKLPKYSLFELENGRKRMLASAGELQKGNE
                     LALPSKYVNFLYLASHYEKLKGSPEDNEQKQLFVEQHKHYLDEIIEQISEFSKRVILAD
                     ANLDKVLSAYNKHRDKPIREQAENIIHLFTLTNLGAPAAFKYFDTTIDRKRYTSTKEVL
                     DATLIHQSITGLYETRIDLSQLGGD"
     CDS             6368..6388
                     /codon_start=1
                     /product="nuclear localization signal of SV40 (simian virus
                     40) large T antigen"
                     /label=SV40 NLS
                     /translation="PKKKRKV"
     CDS             6395..6460
                     /codon_start=1
                     /product="three tandem FLAG(R) epitope tags, followed by an
                     enterokinase cleavage site"
                     /label=3xFLAG
                     /translation="DYKDHDGDYKDHDIDYKDDDDK"
     CDS             6479..6535
                     /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(6536..6538,6539..7252)
                     /codon_start=1
                     /product="the original enhanced GFP (Yang et al., 1996)"
                     /label=EGFP
                     /note="mammalian codon-optimized"
                     /translation="VSKGEELFTGVVPILVELDGDVNGHKFSVSGEGEGDATYGKLTLK
                     FICTTGKLPVPWPTLVTTLTYGVQCFSRYPDHMKQHDFFKSAMPEGYVQERTIFFKDDG
                     NYKTRAEVKFEGDTLVNRIELKGIDFKEDGNILGHKLEYNYNSHNVYIMADKQKNGIKA
                     NFKIRHNIEDGSVQLADHYQQNTPIGDGPVLLPDNHYLSTQSALSKDPNEKRDHMVLLE
                     FVTAAGITLGMDELYK"
     primer_bind     complement(6578..6599)
                     /label=EGFP-N
                     /note="EGFP, reverse primer"
     primer_bind     complement(6839..6858)
                     /label=EXFP-R
                     /note="For distinguishing EGFP variants, reverse primer"
     primer_bind     7186..7207
                     /label=EGFP-C
                     /note="EGFP, forward primer"
     misc_feature    7268..7856
                     /label=WPRE
                     /note="woodchuck hepatitis virus posttranscriptional 
                     regulatory element"
     primer_bind     complement(7321..7341)
                     /label=WPRE-R
                     /note="WPRE, reverse primer"
     CDS             complement(7739..7750)
                     /codon_start=1
                     /product="Factor Xa recognition and cleavage site"
                     /label=Factor Xa site
                     /translation="IEGR"
     promoter        8012..8260
                     /label=U6 promoter
                     /note="RNA polymerase III promoter for human U6 snRNA 
                     (Domitrovich & Kunkel, 2003)"
     primer_bind     8012..8032
                     /label=hU6-F
                     /note="Human U6 promoter, forward primer"
     primer_bind     8183..8202
                     /label=LKO.1 5'
                     /note="Human U6 promoter, forward primer"
     misc_RNA        10142..10217
                     /label=gRNA scaffold
                     /note="guide RNA scaffold for the Streptococcus pyogenes 
                     CRISPR/Cas9 system"
     LTR             10270..10450
                     /label=5' LTR (truncated)
                     /note="truncated 5' long terminal repeat (LTR) from HIV-1"
     polyA_signal    10528..10649
                     /label=SV40 poly(A) signal
                     /note="SV40 polyadenylation signal"
     primer_bind     complement(10565..10584)
                     /label=SV40pA-R
                     /note="SV40 polyA, reverse primer"
     primer_bind     10619..10638
                     /label=EBV-rev
                     /note="SV40 polyA terminator, reverse primer"
     rep_origin      10689..10824
                     /label=SV40 ori
                     /note="SV40 origin of replication"
     primer_bind     complement(10844..10863)
                     /label=T7
                     /note="T7 promoter, forward primer"
     promoter        complement(10845..10863)
                     /label=T7 promoter
                     /note="promoter for bacteriophage T7 RNA polymerase"
     primer_bind     complement(10873..10890)
                     /label=M13 Forward
                     /note="In lacZ gene. Also called M13-F20 or M13 (-21) 
                     Forward"
     primer_bind     complement(10873..10889)
                     /label=M13 fwd
                     /note="common sequencing primer, one of multiple similar 
                     variants"
     primer_bind     complement(10882..10904)
                     /label=M13/pUC Forward
                     /note="In lacZ gene"
     rep_origin      11031..11486
                     /direction=RIGHT
                     /label=f1 ori
                     /note="f1 bacteriophage origin of replication; arrow 
                     indicates direction of (+) strand synthesis"
     primer_bind     complement(11118..11137)
                     /label=F1ori-R
                     /note="F1 origin, reverse primer"
     primer_bind     11328..11349
                     /label=F1ori-F
                     /note="F1 origin, forward primer"
     promoter        11512..11616
                     /gene="bla"
                     /label=AmpR promoter
     CDS             join(11617..11685,11686..12477)
                     /codon_start=1
                     /gene="bla"
                     /product="beta-lactamase"
                     /label=AmpR
                     /note="confers resistance to ampicillin, carbenicillin, and
                     related antibiotics"
                     /translation="MSIQHFRVALIPFFAAFCLPVFAHPETLVKVKDAEDQLGARVGYI
                     ELDLNSGKILESFRPEERFPMMSTFKVLLCGAVLSRIDAGQEQLGRRIHYSQNDLVEYS
                     PVTEKHLTDGMTVRELCSAAITMSDNTAANLLLTTIGGPKELTAFLHNMGDHVTRLDRW
                     EPELNEAIPNDERDTTMPVAMATTLRKLLTGELLTLASRQQLIDWMEADKVAGPLLRSA
                     LPAGWFIADKSGAGERGSRGIIAALGPDGKPSRIVVIYTTGSQATMDERNRQIAEIGAS
                     LIKHW"
     primer_bind     complement(11835..11854)
                     /label=Amp-R
                     /note="Ampicillin resistance gene, reverse primer"
     rep_origin      12648..13236
                     /direction=RIGHT
                     /label=ori
                     /note="high-copy-number ColE1/pMB1/pBR322/pUC origin of 
                     replication"
     primer_bind     13137..13156
                     /label=pBR322ori-F
                     /note="pBR322 origin, forward primer"
     primer_bind     13390..13407
                     /label=L4440
                     /note="L4440 vector, forward primer"
     protein_bind    13524..13545
                     /label=CAP binding site
                     /bound_moiety="E. coli catabolite activator protein"
                     /note="CAP binding activates transcription in the presence 
                     of cAMP."
     promoter        join(13560..13565,13566..13583,13584..13590)
                     /label=lac promoter
                     /note="promoter for the E. coli lac operon"
     protein_bind    13598..13614
                     /label=lac operator
                     /bound_moiety="lac repressor encoded by lacI"
                     /note="The lac repressor binds to the lac operator to 
                     inhibit transcription in E. coli. This inhibition can be 
                     relieved by adding lactose or 
                     isopropyl-beta-D-thiogalactopyranoside (IPTG)."
     primer_bind     13603..13625
                     /label=M13/pUC Reverse
                     /note="In lacZ gene"
     primer_bind     13622..13638
                     /label=M13 rev
                     /note="common sequencing primer, one of multiple similar 
                     variants"
     primer_bind     13622..13638
                     /label=M13 Reverse
                     /note="In lacZ gene. Also called M13-rev"
     primer_bind     13657..13677
                     /label=T3
                     /note="T3 promoter, forward primer"
     promoter        13659..13677
                     /label=T3 promoter
                     /note="promoter for bacteriophage T3 RNA polymerase"
ORIGIN
        1 tgtagtctta tgcaatactc ttgtagtctt gcaacatggt aacgatgagt tagcaacatg
       61 ccttacaagg agagaaaaag caccgtgcat gccgattggt ggaagtaagg tggtacgatc
      121 gtgccttatt aggaaggcaa cagacgggtc tgacatggat tggacgaacc actgaattgc
      181 cgcattgcag agatattgta tttaagtgcc tagctcgata cataaacggg tctctctggt
      241 tagaccagat ctgagcctgg gagctctctg gctaactagg gaacccactg cttaagcctc
      301 aataaagctt gccttgagtg cttcaagtag tgtgtgcccg tctgttgtgt gactctggta
      361 actagagatc cctcagaccc ttttagtcag tgtggaaaat ctctagcagt ggcgcccgaa
      421 cagggacttg aaagcgaaag ggaaaccaga ggagctctct cgacgcagga ctcggcttgc
      481 tgaagcgcgc acggcaagag gcgaggggcg gcgactggtg agtacgccaa aaattttgac
      541 tagcggaggc tagaaggaga gagatgggtg cgagagcgtc agtattaagc gggggagaat
      601 tagatcgcga tgggaaaaaa ttcggttaag gccaggggga aagaaaaaat ataaattaaa
      661 acatatagta tgggcaagca gggagctaga acgattcgca gttaatcctg gcctgttaga
      721 aacatcagaa ggctgtagac aaatactggg acagctacaa ccatcccttc agacaggatc
      781 agaagaactt agatcattat ataatacagt agcaaccctc tattgtgtgc atcaaaggat
      841 agagataaaa gacaccaagg aagctttaga caagatagag gaagagcaaa acaaaagtaa
      901 gaccaccgca cagcaagcgg ccgctgatct tcagacctgg aggaggagat atgagggaca
      961 attggagaag tgaattatat aaatataaag tagtaaaaat tgaaccatta ggagtagcac
     1021 ccaccaaggc aaagagaaga gtggtgcaga gagaaaaaag agcagtggga ataggagctt
     1081 tgttccttgg gttcttggga gcagcaggaa gcactatggg cgcagcgtca atgacgctga
     1141 cggtacaggc cagacaatta ttgtctggta tagtgcagca gcagaacaat ttgctgaggg
     1201 ctattgaggc gcaacagcat ctgttgcaac tcacagtctg gggcatcaag cagctccagg
     1261 caagaatcct ggctgtggaa agatacctaa aggatcaaca gctcctgggg atttggggtt
     1321 gctctggaaa actcatttgc accactgctg tgccttggaa tgctagttgg agtaataaat
     1381 ctctggaaca gatttggaat cacacgacct ggatggagtg ggacagagaa attaacaatt
     1441 acacaagctt aatacactcc ttaattgaag aatcgcaaaa ccagcaagaa aagaatgaac
     1501 aagaattatt ggaattagat aaatgggcaa gtttgtggaa ttggtttaac ataacaaatt
     1561 ggctgtggta tataaaatta ttcataatga tagtaggagg cttggtaggt ttaagaatag
     1621 tttttgctgt actttctata gtgaatagag ttaggcaggg atattcacca ttatcgtttc
     1681 agaatcttga gacaaatggc agtattcatc cacaatttta aaagaaaagg ggggattggg
     1741 gggtacagtg caggggaaag aatagtagac ataatagcaa cagacataca aactaaagaa
     1801 ttacaaaaac aaattacaaa aattcaaaat tttcgggttt attacaggga cagcagagat
     1861 ccactttggc gccggctcga gggggcccgg gtgcaaagat ggataaagtt ttaaacagag
     1921 aggaatcttt gcagctaatg gaccttctag gtcttgaaag gagtgggaat tggctccgta
     1981 ggtcttgaaa ggagtgggaa ttggctccgg tgcccgtcag tgggcagagc gcacatcgcc
     2041 cacagtcccc gagaagttgg ggggaggggt cggcaattga tccggtgcct agagaaggtg
     2101 gcgcggggta aactgggaaa gtgatgtcgt gtactggctc cgcctttttc ccgagggtgg
     2161 gggagaaccg tatataagtg cagtagtcgc cgtgaacgtt ctttttcgca acgggtttgc
     2221 cgccagaaca caggaccggt tctagagcgc tgccaccatg gataaaaagt attctattgg
     2281 tttagacatc ggcactaatt ccgttggatg ggctgtcata accgatgaat acaaagtacc
     2341 ttcaaagaaa tttaaggtgt tggggaacac agaccgtcat tcgattaaaa agaatcttat
     2401 cggtgccctc ctattcgata gtggcgaaac ggcagaggcg actcgcctga aacgaaccgc
     2461 tcggagaagg tatacacgtc gcaagaaccg aatatgttac ttacaagaaa tttttagcaa
     2521 tgagatggcc aaagttgacg attctttctt tcaccgtttg gaagagtcct tccttgtcga
     2581 agaggacaag aaacatgaac ggcaccccat ctttggaaac atagtagatg aggtggcata
     2641 tcatgaaaag tacccaacga tttatcacct cagaaaaaag ctagttgact caactgataa
     2701 agcggacctg aggttaatct acttggctct tgcccatatg ataaagttcc gtgggcactt
     2761 tctcattgag ggtgatctaa atccggacaa ctcggatgtc gacaaactgt tcatccagtt
     2821 agtacaaacc tataatcagt tgtttgaaga gaaccctata aatgcaagtg gcgtggatgc
     2881 gaaggctatt cttagcgccc gcctctctaa atcccgacgg ctagaaaacc tgatcgcaca
     2941 attacccgga gagaagaaaa atgggttgtt cggtaacctt atagcgctct cactaggcct
     3001 gacaccaaat tttaagtcga acttcgactt agctgaagat gccaaattgc agcttagtaa
     3061 ggacacgtac gatgacgatc tcgacaatct actggcacaa attggagatc agtatgcgga
     3121 cttatttttg gctgccaaaa accttagcga tgcaatcctc ctatctgaca tactgagagt
     3181 taatactgag attaccaagg cgccgttatc cgcttcaatg atcaaaaggt acgatgaaca
     3241 tcaccaagac ttgacacttc tcaaggccct agtccgtcag caactgcctg agaaatataa
     3301 ggaaatattc tttgatcagt cgaaaaacgg gtacgcaggt tatattgacg gcggagcgag
     3361 tcaagaggaa ttctacaagt ttatcaaacc catattagag aagatggatg ggacggaaga
     3421 gttgcttgta aaactcaatc gcgaagatct actgcgaaag cagcggactt tcgacaacgg
     3481 tagcattcca catcaaatcc acttaggcga attgcatgct atacttagaa ggcaggagga
     3541 tttttatccg ttcctcaaag acaatcgtga aaagattgag aaaatcctaa cctttcgcat
     3601 accttactat gtgggacccc tggcccgagg gaactctcgg ttcgcatgga tgacaagaaa
     3661 gtccgaagaa acgattactc catggaattt tgaggaagtt gtcgataaag gtgcgtcagc
     3721 tcaatcgttc atcgagagga tgaccaactt tgacaagaat ttaccgaacg aaaaagtatt
     3781 gcctaagcac agtttacttt acgagtattt cacagtgtac aatgaactca cgaaagttaa
     3841 gtatgtcact gagggcatgc gtaaacccgc ctttctaagc ggagaacaga agaaagcaat
     3901 agtagatctg ttattcaaga ccaaccgcaa agtgacagtt aagcaattga aagaggacta
     3961 ctttaagaaa attgaatgct tcgattctgt cgagatctcc ggggtagaag atcgatttaa
     4021 tgcgtcactt ggtacgtatc atgacctcct aaagataatt aaagataagg acttcctgga
     4081 taacgaagag aatgaagata tcttagaaga tatagtgttg actcttaccc tctttgaaga
     4141 tcgggaaatg attgaggaaa gactaaaaac atacgctcac ctgttcgacg ataaggttat
     4201 gaaacagtta aagaggcgtc gctatacggg ctggggacga ttgtcgcgga aacttatcaa
     4261 cgggataaga gacaagcaaa gtggtaaaac tattctcgat tttctaaaga gcgacggctt
     4321 cgccaatagg gcctttgcag ccctgatcgc tgatgactct ttaaccttca aagaggatat
     4381 acaaaaggca caggtttccg gacaagggga ctcattgcac gaacatattg cgaatcttgc
     4441 tggttcgcca gccatcaaaa agggcatact ccagacagtc aaagtagtgg atgagctagt
     4501 taaggtcatg ggacgtcaca aaccggaaaa cattgtaatc gagatggcac gcgaaaatca
     4561 aacgactcag aaggggcaaa aaaacagtcg agagcggatg aagagaatag aagagggtat
     4621 taaagaactg ggcagccaga tcttaaagga gcatcctgtg gaaaataccc aattgcagaa
     4681 cgagaaactt tacctctatt acctacaaaa tggaagggac atgtatgttg atcaggaact
     4741 ggacataaac cgtttatctg attacgacgt cgatcacatt gtaccccaat cctttttgaa
     4801 ggacgattca atcgacaata aagtgcttac acgctcggat aagaaccgag ggaaaagtga
     4861 caatgttcca agcgaggaag tcgtaaagaa aatgaagaac tattggcggc agctcctaaa
     4921 tgcgaaactg ataacgcaaa gaaagttcga taacttaact aaagctgaga ggggtggctt
     4981 gtctgaactt gacaaggccg gatttattaa acgtcagctc gtggaaaccc gccaaatcac
     5041 aaagcatgtt gcacagatac tagattcccg aatgaatacg aaatacgacg agaacgataa
     5101 gctgattcgg gaagtcaaag taatcacttt aaagtcaaaa ttggtgtcgg acttcagaaa
     5161 ggattttcaa ttctataaag ttagggagat aaataactac caccatgcgc acgacgctta
     5221 tcttaatgcc gtcgtaggga ccgcactcat taagaaatac ccgaagctag aaagtgagtt
     5281 tgtgtatggt gattacaaag tttatgacgt ccgtaagatg atcgcgaaaa gcgaacagga
     5341 gataggcaag gctacagcca aatacttctt ttattctaac attatgaatt tctttaagac
     5401 ggaaatcact ctggcaaacg gagagatacg caaacgacct ttaattgaaa ccaatgggga
     5461 gacaggtgaa atcgtatggg ataagggccg ggacttcgcg acggtgagaa aagttttgtc
     5521 catgccccaa gtcaacatag taaagaaaac tgaggtgcag accggagggt tttcaaagga
     5581 atcgattctt ccaaaaagga atagtgataa gctcatcgct cgtaaaaagg actgggaccc
     5641 gaaaaagtac ggtggcttcg atagccctac agttgcctat tctgtcctag tagtggcaaa
     5701 agttgagaag ggaaaatcca agaaactgaa gtcagtcaaa gaattattgg ggataacgat
     5761 tatggagcgc tcgtcttttg aaaagaaccc catcgacttc cttgaggcga aaggttacaa
     5821 ggaagtaaaa aaggatctca taattaaact accaaagtat agtctgtttg agttagaaaa
     5881 tggccgaaaa cggatgttgg ctagcgccgg agagcttcaa aaggggaacg aactcgcact
     5941 accgtctaaa tacgtgaatt tcctgtattt agcgtcccat tacgagaagt tgaaaggttc
     6001 acctgaagat aacgaacaga agcaactttt tgttgagcag cacaaacatt atctcgacga
     6061 aatcatagag caaatttcgg aattcagtaa gagagtcatc ctagctgatg ccaatctgga
     6121 caaagtatta agcgcataca acaagcacag ggataaaccc atacgtgagc aggcggaaaa
     6181 tattatccat ttgtttactc ttaccaacct cggcgctcca gccgcattca agtattttga
     6241 cacaacgata gatcgcaaac gatacacttc taccaaggag gtgctagacg cgacactgat
     6301 tcaccaatcc atcacgggat tatatgaaac tcggatagat ttgtcacagc ttgggggtga
     6361 cggatccccc aagaagaaga ggaaagtctc gagcgactac aaagaccatg acggtgatta
     6421 taaagatcat gacatcgatt acaaggatga cgatgacaag gctgcaggag gatccggcgc
     6481 aacaaacttc tctctgctga aacaagccgg agatgtcgaa gagaatcctg gaccggtgag
     6541 caagggcgag gagctgttca ccggggtggt gcccatcctg gtcgagctgg acggcgacgt
     6601 aaacggccac aagttcagcg tgtctggcga gggcgagggc gatgccacct acggcaagct
     6661 gaccctgaag ttcatctgca ccaccggcaa gctgcccgtg ccctggccca ccctcgtgac
     6721 caccctgacc tacggcgtgc agtgcttcag ccgctacccc gaccacatga agcagcacga
     6781 cttcttcaag tccgccatgc ccgaaggcta cgtccaggag cgcaccatct tcttcaagga
     6841 cgacggcaac tacaagaccc gcgccgaggt gaagttcgag ggcgacaccc tggtgaaccg
     6901 catcgagctg aagggcatcg acttcaagga ggacggcaac atcctggggc acaagctgga
     6961 gtacaactac aacagccaca acgtctatat catggccgac aagcagaaga acggcatcaa
     7021 ggcgaacttc aagatccgcc acaacatcga ggacggcagc gtgcagctcg ccgaccacta
     7081 ccagcagaac acccccatcg gcgacggccc cgtgctgctg cccgacaacc actacctgag
     7141 cacccagtcc gccctgagca aagaccccaa cgagaagcgc gatcacatgg tcctgctgga
     7201 gttcgtgacc gccgccggga tcactctcgg catggacgag ctgtacaagt aaacgcgtta
     7261 agtcgacaat caacctctgg attacaaaat ttgtgaaaga ttgactggta ttcttaacta
     7321 tgttgctcct tttacgctat gtggatacgc tgctttaatg cctttgtatc atgctattgc
     7381 ttcccgtatg gctttcattt tctcctcctt gtataaatcc tggttgctgt ctctttatga
     7441 ggagttgtgg cccgttgtca ggcaacgtgg cgtggtgtgc actgtgtttg ctgacgcaac
     7501 ccccactggt tggggcattg ccaccacctg tcagctcctt tccgggactt tcgctttccc
     7561 cctccctatt gccacggcgg aactcatcgc cgcctgcctt gcccgctgct ggacaggggc
     7621 tcggctgttg ggcactgaca attccgtggt gttgtcgggg aaatcatcgt cctttccttg
     7681 gctgctcgcc tgtgttgcca cctggattct gcgcgggacg tccttctgct acgtcccttc
     7741 ggccctcaat ccagcggacc ttccttcccg cggcctgctg ccggctctgc ggcctcttcc
     7801 gcgtcttcgc cttcgccctc agacgagtcg gatctccctt tgggccgcct ccccgcgtcg
     7861 actttaagac caatgactta caaggcagct gtagatctta gccacttttt aaaagaaaag
     7921 gggggactgg aagggctaat tcactcccaa cgaagacaag atttcaccat tatcgtttca
     7981 gacccacctc ccaaccccga ggggacccag agagggccta tttcccatga ttccttcata
     8041 tttgcatata cgatacaagg ctgttagaga gataattaga attaatttga ctgtaaacac
     8101 aaagatatta gtacaaaata cgtgacgtag aaagtaataa tttcttgggt agtttgcagt
     8161 tttaaaatta tgttttaaaa tggactatca tatgcttacc gtaacttgaa agtatttcga
     8221 tttcttggct ttatatatct tgtggaaagg acgaaacacc ggagacggtt gtaaatgagc
     8281 acacaaaata cacatgctaa aatattatat tctatgacct ttataaaatc aaccaaaatc
     8341 ttctttttaa taactttagt atcaataatt agaattttta tgttcctttt tgcaaacttt
     8401 taataaaaat gagcaaaata aaaaaacgct agttttagta actcgcgttg ttttcttcac
     8461 ctttaataat agctactcca ccacttgttc ctaagcggtc agctcctgct tcaatcattt
     8521 tttgagcatc ttcaaatgtt ctaactccac cagctgcttt aactaaagca ttgtctttaa
     8581 caactgactt cattagttta acatcttcaa atgttgcacc tgattttgaa aatcctgttg
     8641 atgttttaac aaattctaat ccagcttcaa cagctatttc acaagctttc atgatttctt
     8701 cttttgttaa taaacaattt tccataatac atttaacaac atgtgatcca gctgcttttt
     8761 ttacagcttt catgtcttct aaaactaatt cataattttt gtcttttaat gcaccaatat
     8821 ttaataccat atcaatttct gttgcaccat ctttaattgc ttcagaaact tcgaatgctt
     8881 ttgtagctgt tgtgcatgca cctagaggaa aacctacaac atttgttatt cctacatttg
     8941 tgccttttaa taattcttta caatagcttg ttcaatatga attaacacaa actgttgcaa
     9001 aatcaaattc aattgcttca tcacataatt gtttaatttc agctttcgta gcatcttgtt
     9061 ttaataatgt gtgatctata tatttgttta gtttcatttt ttctcctata tattcatttt
     9121 taattttaat tctttaataa tttcgtctac tttaacttta gcgttttgaa cagattcacc
     9181 aacacctata aaataaattt ttagtttagg ttcagttcca cttgggcgaa cagcaaatca
     9241 tgacttatct tctaaataaa attttagtaa gtcttgtcct ggcatattat acattccatc
     9301 gatgtagtct tcaacattaa caactttaag tccagcaatt tgagttaagg gtgttgctct
     9361 caatgatttc attaatggtt caatttttaa tttcttttct tctggtttaa aattcaagtt
     9421 taaagtgaaa gtgtaatatg cacccatttc tttaaataaa tcttctaaat agtctactaa
     9481 tgttttattt tgttttttat aaaatcaagc agcctctgct attaatatag aagcttgtat
     9541 tccatcttta tctctagctg agtcatcaat tacatatcca taactttctt cataagcaaa
     9601 aacaaaattt aatccgttat cttcttcttt agcaatttct ctacccattc atttaaatcc
     9661 agttaaagtt tttacaatat taactccata tttttcatga gcgattctat cacccaaatc
     9721 acttgttaca aaacttgaat atagagccgg attttttgga atgctattta agcgttttag
     9781 atttgataat tttcaatcaa ttaaaattgg tcctgtttga tttccatcta atcttacaaa
     9841 atgaccatca tgttttattg ccattccaaa tctgtcagca tctgggtcat tcataataat
     9901 aatatctgca tcatgtttaa taccatattc aagcggtatt tttcatgcag gatcaaattc
     9961 tggatttgga tttacaacat ttttaaatgt ttcatcttca aatgcatgct cttcaacctc
    10021 aataacgtta tatcctgatt cacgtaatat ttttggggta aatttagttc ctgttccatt
    10081 aactgcgcta aaaataattt ttaaatcttt tttagcttct tgctcttttt tgtacgtctc
    10141 tgttttagag ctagaaatag caagttaaaa taaggctagt ccgttatcaa cttgaaaaag
    10201 tggcaccgag tcggtgcttt tttaagcttg gcgtaactag atcttgagac actgcttttt
    10261 gcttgtactg ggtctctctg gttagaccag atctgagcct gggagctctc tggctaacta
    10321 gggaacccac tgcttaagcc tcaataaagc ttgccttgag tgcttcaagt agtgtgtgcc
    10381 cgtctgttgt gtgactctgg taactagaga tccctcagac ccttttagtc agtgtggaaa
    10441 atctctagca gtacgtatag tagttcatgt catcttatta ttcagtattt ataacttgca
    10501 aagaaatgaa tatcagagag tgagaggaac ttgtttattg cagcttataa tggttacaaa
    10561 taaagcaata gcatcacaaa tttcacaaat aaagcatttt tttcactgca ttctagttgt
    10621 ggtttgtcca aactcatcaa tgtatcttat catgtctggc tctagctatc ccgcccctaa
    10681 ctccgcccat cccgccccta actccgccca gttccgccca ttctccgccc catggctgac
    10741 taattttttt tatttatgca gaggccgagg ccgcctcggc ctctgagcta ttccagaagt
    10801 agtgaggagg cttttttgga ggcctaggga cgtacccaat tcgccctata gtgagtcgta
    10861 ttacgcgcgc tcactggccg tcgttttaca acgtcgtgac tgggaaaacc ctggcgttac
    10921 ccaacttaat cgccttgcag cacatccccc tttcgccagc tggcgtaata gcgaagaggc
    10981 ccgcaccgat cgcccttccc aacagttgcg cagcctgaat ggcgaatggg acgcgccctg
    11041 tagcggcgca ttaagcgcgg cgggtgtggt ggttacgcgc agcgtgaccg ctacacttgc
    11101 cagcgcccta gcgcccgctc ctttcgcttt cttcccttcc tttctcgcca cgttcgccgg
    11161 ctttccccgt caagctctaa atcgggggct ccctttaggg ttccgattta gtgctttacg
    11221 gcacctcgac cccaaaaaac ttgattaggg tgatggttca cgtagtgggc catcgccctg
    11281 atagacggtt tttcgccctt tgacgttgga gtccacgttc tttaatagtg gactcttgtt
    11341 ccaaactgga acaacactca accctatctc ggtctattct tttgatttat aagggatttt
    11401 gccgatttcg gcctattggt taaaaaatga gctgatttaa caaaaattta acgcgaattt
    11461 taacaaaata ttaacgctta caatttaggt ggcacttttc ggggaaatgt gcgcggaacc
    11521 cctatttgtt tatttttcta aatacattca aatatgtatc cgctcatgag acaataaccc
    11581 tgataaatgc ttcaataata ttgaaaaagg aagagtatga gtattcaaca tttccgtgtc
    11641 gcccttattc ccttttttgc ggcattttgc cttcctgttt ttgctcaccc agaaacgctg
    11701 gtgaaagtaa aagatgctga agatcagttg ggtgcacgag tgggttacat cgaactggat
    11761 ctcaacagcg gtaagatcct tgagagtttt cgccccgaag aacgttttcc aatgatgagc
    11821 acttttaaag ttctgctatg tggcgcggta ttatcccgta ttgacgccgg gcaagagcaa
    11881 ctcggtcgcc gcatacacta ttctcagaat gacttggttg agtactcacc agtcacagaa
    11941 aagcatctta cggatggcat gacagtaaga gaattatgca gtgctgccat aaccatgagt
    12001 gataacactg cggccaactt acttctgaca acgatcggag gaccgaagga gctaaccgct
    12061 tttttgcaca acatggggga tcatgtaact cgccttgatc gttgggaacc ggagctgaat
    12121 gaagccatac caaacgacga gcgtgacacc acgatgcctg tagcaatggc aacaacgttg
    12181 cgcaaactat taactggcga actacttact ctagcttccc ggcaacaatt aatagactgg
    12241 atggaggcgg ataaagttgc aggaccactt ctgcgctcgg cccttccggc tggctggttt
    12301 attgctgata aatctggagc cggtgagcgt gggtctcgcg gtatcattgc agcactgggg
    12361 ccagatggta agccctcccg tatcgtagtt atctacacga cggggagtca ggcaactatg
    12421 gatgaacgaa atagacagat cgctgagata ggtgcctcac tgattaagca ttggtaactg
    12481 tcagaccaag tttactcata tatactttag attgatttaa aacttcattt ttaatttaaa
    12541 aggatctagg tgaagatcct ttttgataat ctcatgacca aaatccctta acgtgagttt
    12601 tcgttccact gagcgtcaga ccccgtagaa aagatcaaag gatcttcttg agatcctttt
    12661 tttctgcgcg taatctgctg cttgcaaaca aaaaaaccac cgctaccagc ggtggtttgt
    12721 ttgccggatc aagagctacc aactcttttt ccgaaggtaa ctggcttcag cagagcgcag
    12781 ataccaaata ctgttcttct agtgtagccg tagttaggcc accacttcaa gaactctgta
    12841 gcaccgccta catacctcgc tctgctaatc ctgttaccag tggctgctgc cagtggcgat
    12901 aagtcgtgtc ttaccgggtt ggactcaaga cgatagttac cggataaggc gcagcggtcg
    12961 ggctgaacgg ggggttcgtg cacacagccc agcttggagc gaacgaccta caccgaactg
    13021 agatacctac agcgtgagct atgagaaagc gccacgcttc ccgaagggag aaaggcggac
    13081 aggtatccgg taagcggcag ggtcggaaca ggagagcgca cgagggagct tccaggggga
    13141 aacgcctggt atctttatag tcctgtcggg tttcgccacc tctgacttga gcgtcgattt
    13201 ttgtgatgct cgtcaggggg gcggagccta tggaaaaacg ccagcaacgc ggccttttta
    13261 cggttcctgg ccttttgctg gccttttgct cacatgttct ttcctgcgtt atcccctgat
    13321 tctgtggata accgtattac cgcctttgag tgagctgata ccgctcgccg cagccgaacg
    13381 accgagcgca gcgagtcagt gagcgaggaa gcggaagagc gcccaatacg caaaccgcct
    13441 ctccccgcgc gttggccgat tcattaatgc agctggcacg acaggtttcc cgactggaaa
    13501 gcgggcagtg agcgcaacgc aattaatgtg agttagctca ctcattaggc accccaggct
    13561 ttacacttta tgcttccggc tcgtatgttg tgtggaattg tgagcggata acaatttcac
    13621 acaggaaaca gctatgacca tgattacgcc aagcgcgcaa ttaaccctca ctaaagggaa
    13681 caaaagctgg agctgcaagc ttaa
//
