LOCUS       sequence-374759-       15055 bp    DNA     circular SYN 15-DEC-2025
DEFINITION  dCas9 with an N-term Zim3 KRAB-NLS fusion, a C-term HA-2xNLS.
ACCESSION   .
VERSION     .
KEYWORDS    .
SOURCE      synthetic DNA construct
  ORGANISM  synthetic DNA construct
REFERENCE   1  (bases 1 to 15055)
  AUTHORS   Replogle JM, Bonnar JL, Pogson AN, Liem CR, Maier NK, Ding Y, 
            Russell BJ, Wang X, Leng K, Guna A, Norman TM, Pak RA, Ramos DM, 
            Ward ME, Gilbert LA, Kampmann M, Weissman JS, Jost M
  TITLE     Maximizing CRISPRi efficacy and accessibility with dual-sgRNA 
            libraries and optimal effectors.
  JOURNAL   eLife. 2022 Dec 28;11:e81856. doi: 10.7554/eLife.81856.
  PUBMED    36576240
REFERENCE   2  (bases 1 to 15055)
  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: "eLife."; 
            date: "2022-12-28"; volume: "11:e81856. doi"; pages: " 
            10.7554/eLife.81856"
COMMENT     Sequence Label: pHR-UCOE-SFFV-Zim3-dCas9-loxP-P2A-EGFP-loxP
FEATURES             Location/Qualifiers
     source          1..15055
                     /mol_type="other DNA"
                     /organism="synthetic DNA construct"
     primer_bind     complement(38..58)
                     /label=pBABE 3'
                     /note="SV40 enhancer, reverse primer for pBABE vectors"
     promoter        43..372
                     /label=SV40 promoter
                     /note="SV40 enhancer and early promoter"
     rep_origin      223..358
                     /label=SV40 ori
                     /note="SV40 origin of replication"
     intron          1506..1571
                     /label=small t intron
                     /note="SV40 (simian virus 40) small t antigen intron"
     CDS             1701..1721
                     /codon_start=1
                     /product="nuclear localization signal of SV40 (simian virus
                     40) large T antigen"
                     /label=SV40 NLS
                     /translation="PKKKRKV"
     polyA_signal    2146..2280
                     /label=SV40 poly(A) signal
                     /note="SV40 polyadenylation signal"
     primer_bind     complement(2183..2202)
                     /label=SV40pA-R
                     /note="SV40 polyA, reverse primer"
     primer_bind     2237..2256
                     /label=EBV-rev
                     /note="SV40 polyA terminator, reverse primer"
     LTR             2448..3081
                     /label=3' LTR
                     /note="3' long terminal repeat (LTR) from HIV-1"
     misc_feature    3128..3253
                     /label=HIV-1 Psi
                     /note="packaging signal of human immunodeficiency virus 
                     type 1"
     misc_feature    3750..3983
                     /label=RRE
                     /note="The Rev response element (RRE) of HIV-1 allows for 
                     Rev-dependent mRNA export from the nucleus to the 
                     cytoplasm."
     CDS             4168..4212
                     /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    4497..4614
                     /label=cPPT/CTS
                     /note="central polypurine tract and central termination 
                     sequence of HIV-1"
     promoter        5356..5763
                     /label=SFFV promoter
                     /note="spleen focus-forming virus long terminal repeat 
                     (LTR) promoter"
     protein_bind    complement(6139..6163)
                     /gene="mutant version of attB"
                     /label=attB2
                     /bound_moiety="BP Clonase(TM)"
                     /note="recombination site for the Gateway(R) BP reaction"
     CDS             6183..6203
                     /codon_start=1
                     /product="nuclear localization signal of SV40 (simian virus
                     40) large T antigen"
                     /label=SV40 NLS
                     /translation="PKKKRKV"
     regulatory      6252..6261
                     /label=Kozak sequence
                     /note="vertebrate consensus sequence for strong initiation 
                     of translation (Kozak, 1987)"
                     /regulatory_class="other"
     CDS             join(6258..6284,6285..6287,6288..8774,8775..8777,
                     8778..10361)
                     /codon_start=1
                     /product="catalytically dead mutant of the Cas9 
                     endonuclease from the Streptococcus pyogenes Type II 
                     CRISPR/Cas system"
                     /label=dCas9
                     /note="RNA-guided DNA-binding protein that lacks 
                     endonuclease activity due to the D10A mutation in the RuvC 
                     catalytic domain and the H840A mutation in the HNH 
                     catalytic domain"
                     /translation="MDKKYSIGLAIGTNSVGWAVITDEYKVPSKKFKVLGNTDRHSIKK
                     NLIGALLFDSGETAEATRLKRTARRRYTRRKNRICYLQEIFSNEMAKVDDSFFHRLEES
                     FLVEEDKKHERHPIFGNIVDEVAYHEKYPTIYHLRKKLVDSTDKADLRLIYLALAHMIK
                     FRGHFLIEGDLNPDNSDVDKLFIQLVQTYNQLFEENPINASGVDAKAILSARLSKSRRL
                     ENLIAQLPGEKKNGLFGNLIALSLGLTPNFKSNFDLAEDAKLQLSKDTYDDDLDNLLAQ
                     IGDQYADLFLAAKNLSDAILLSDILRVNTEITKAPLSASMIKRYDEHHQDLTLLKALVR
                     QQLPEKYKEIFFDQSKNGYAGYIDGGASQEEFYKFIKPILEKMDGTEELLVKLNREDLL
                     RKQRTFDNGSIPHQIHLGELHAILRRQEDFYPFLKDNREKIEKILTFRIPYYVGPLARG
                     NSRFAWMTRKSEETITPWNFEEVVDKGASAQSFIERMTNFDKNLPNEKVLPKHSLLYEY
                     FTVYNELTKVKYVTEGMRKPAFLSGEQKKAIVDLLFKTNRKVTVKQLKEDYFKKIECFD
                     SVEISGVEDRFNASLGTYHDLLKIIKDKDFLDNEENEDILEDIVLTLTLFEDREMIEER
                     LKTYAHLFDDKVMKQLKRRRYTGWGRLSRKLINGIRDKQSGKTILDFLKSDGFANRNFM
                     QLIHDDSLTFKEDIQKAQVSGQGDSLHEHIANLAGSPAIKKGILQTVKVVDELVKVMGR
                     HKPENIVIEMARENQTTQKGQKNSRERMKRIEEGIKELGSQILKEHPVENTQLQNEKLY
                     LYYLQNGRDMYVDQELDINRLSDYDVDAIVPQSFLKDDSIDNKVLTRSDKNRGKSDNVP
                     SEEVVKKMKNYWRQLLNAKLITQRKFDNLTKAERGGLSELDKAGFIKRQLVETRQITKH
                     VAQILDSRMNTKYDENDKLIREVKVITLKSKLVSDFRKDFQFYKVREINNYHHAHDAYL
                     NAVVGTALIKKYPKLESEFVYGDYKVYDVRKMIAKSEQEIGKATAKYFFYSNIMNFFKT
                     EITLANGEIRKRPLIETNGETGEIVWDKGRDFATVRKVLSMPQVNIVKKTEVQTGGFSK
                     ESILPKRNSDKLIARKKDWDPKKYGGFDSPTVAYSVLVVAKVEKGKSKKLKSVKELLGI
                     TIMERSSFEKNPIDFLEAKGYKEVKKDLIIKLPKYSLFELENGRKRMLASAGELQKGNE
                     LALPSKYVNFLYLASHYEKLKGSPEDNEQKQLFVEQHKHYLDEIIEQISEFSKRVILAD
                     ANLDKVLSAYNKHRDKPIREQAENIIHLFTLTNLGAPAAFKYFDTTIDRKRYTSTKEVL
                     DATLIHQSITGLYETRIDLSQLGGD"
     CDS             6258..10361
                     /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="MDKKYSIGLAIGTNSVGWAVITDEYKVPSKKFKVLGNTDRHSIKK
                     NLIGALLFDSGETAEATRLKRTARRRYTRRKNRICYLQEIFSNEMAKVDDSFFHRLEES
                     FLVEEDKKHERHPIFGNIVDEVAYHEKYPTIYHLRKKLVDSTDKADLRLIYLALAHMIK
                     FRGHFLIEGDLNPDNSDVDKLFIQLVQTYNQLFEENPINASGVDAKAILSARLSKSRRL
                     ENLIAQLPGEKKNGLFGNLIALSLGLTPNFKSNFDLAEDAKLQLSKDTYDDDLDNLLAQ
                     IGDQYADLFLAAKNLSDAILLSDILRVNTEITKAPLSASMIKRYDEHHQDLTLLKALVR
                     QQLPEKYKEIFFDQSKNGYAGYIDGGASQEEFYKFIKPILEKMDGTEELLVKLNREDLL
                     RKQRTFDNGSIPHQIHLGELHAILRRQEDFYPFLKDNREKIEKILTFRIPYYVGPLARG
                     NSRFAWMTRKSEETITPWNFEEVVDKGASAQSFIERMTNFDKNLPNEKVLPKHSLLYEY
                     FTVYNELTKVKYVTEGMRKPAFLSGEQKKAIVDLLFKTNRKVTVKQLKEDYFKKIECFD
                     SVEISGVEDRFNASLGTYHDLLKIIKDKDFLDNEENEDILEDIVLTLTLFEDREMIEER
                     LKTYAHLFDDKVMKQLKRRRYTGWGRLSRKLINGIRDKQSGKTILDFLKSDGFANRNFM
                     QLIHDDSLTFKEDIQKAQVSGQGDSLHEHIANLAGSPAIKKGILQTVKVVDELVKVMGR
                     HKPENIVIEMARENQTTQKGQKNSRERMKRIEEGIKELGSQILKEHPVENTQLQNEKLY
                     LYYLQNGRDMYVDQELDINRLSDYDVDAIVPQSFLKDDSIDNKVLTRSDKNRGKSDNVP
                     SEEVVKKMKNYWRQLLNAKLITQRKFDNLTKAERGGLSELDKAGFIKRQLVETRQITKH
                     VAQILDSRMNTKYDENDKLIREVKVITLKSKLVSDFRKDFQFYKVREINNYHHAHDAYL
                     NAVVGTALIKKYPKLESEFVYGDYKVYDVRKMIAKSEQEIGKATAKYFFYSNIMNFFKT
                     EITLANGEIRKRPLIETNGETGEIVWDKGRDFATVRKVLSMPQVNIVKKTEVQTGGFSK
                     ESILPKRNSDKLIARKKDWDPKKYGGFDSPTVAYSVLVVAKVEKGKSKKLKSVKELLGI
                     TIMERSSFEKNPIDFLEAKGYKEVKKDLIIKLPKYSLFELENGRKRMLASAGELQKGNE
                     LALPSKYVNFLYLASHYEKLKGSPEDNEQKQLFVEQHKHYLDEIIEQISEFSKRVILAD
                     ANLDKVLSAYNKHRDKPIREQAENIIHLFTLTNLGAPAAFKYFDTTIDRKRYTSTKEVL
                     DATLIHQSITGLYETRIDLSQLGGD"
     CDS             10365..10391
                     /codon_start=1
                     /product="HA (human influenza hemagglutinin) epitope tag"
                     /label=HA
                     /translation="YPYDVPDYA"
     CDS             10371..10397
                     /codon_start=1
                     /product="HA (human influenza hemagglutinin) epitope tag"
                     /label=HA
                     /note="This is a variant of the standard HA tag, which has 
                     sequence YPYDVPDYA. Some anti-HA antibodies may not 
                     recognize this variant."
                     /translation="YDVPDYASL"
     CDS             10410..10430
                     /codon_start=1
                     /product="nuclear localization signal of SV40 (simian virus
                     40) large T antigen"
                     /label=SV40 NLS
                     /translation="PKKKRKV"
     CDS             10437..10457
                     /codon_start=1
                     /product="nuclear localization signal of SV40 (simian virus
                     40) large T antigen"
                     /label=SV40 NLS
                     /translation="PKKKRKV"
     protein_bind    10467..10500
                     /label=lox2272
                     /bound_moiety="Cre recombinase"
                     /note="Cre-mediated recombination occurs in the 8-bp core 
                     sequence (AAGTATCC) (Shaw et al., 2021). lox2272 sites are 
                     compatible with each other, but incompatible with loxP or 
                     loxN sites (Lee and Saito, 1988)."
     CDS             10503..10559
                     /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(10560..10562,10563..11276)
                     /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(10863..10882)
                     /label=EXFP-R
                     /note="For distinguishing EGFP variants, reverse primer"
     primer_bind     11210..11231
                     /label=EGFP-C
                     /note="EGFP, forward primer"
     protein_bind    11276..11309
                     /label=lox2272
                     /bound_moiety="Cre recombinase"
                     /note="Cre-mediated recombination occurs in the 8-bp core 
                     sequence (AAGTATCC) (Shaw et al., 2021). lox2272 sites are 
                     compatible with each other, but incompatible with loxP or 
                     loxN sites (Lee and Saito, 1988)."
     misc_feature    11366..11954
                     /label=WPRE
                     /note="woodchuck hepatitis virus posttranscriptional 
                     regulatory element"
     primer_bind     complement(11419..11439)
                     /label=WPRE-R
                     /note="WPRE, reverse primer"
     CDS             complement(11837..11848)
                     /codon_start=1
                     /product="Factor Xa recognition and cleavage site"
                     /label=Factor Xa site
                     /translation="IEGR"
     primer_bind     complement(11957..11973)
                     /label=KS primer
                     /note="common sequencing primer, one of multiple similar 
                     variants"
     primer_bind     complement(11958..11974)
                     /label=pBluescriptKS
                     /note="For pBluescript vector"
     LTR             12064..12297
                     /label=3' LTR (Delta-U3)
                     /note="self-inactivating 3' long terminal repeat (LTR) from
                     HIV-1"
     promoter        complement(12322..12340)
                     /label=SP6 promoter
                     /note="promoter for bacteriophage SP6 RNA polymerase"
     primer_bind     complement(12323..12340)
                     /label=SP6
                     /note="SP6 promoter, forward primer"
     primer_bind     complement(12630..12649)
                     /label=pBRrevBam
                     /note="pBR322 vectors, tet region, downstream of BamHI, 
                     reverse primer"
     primer_bind     complement(12863..12882)
                     /label=pRS-marker
                     /note="pRS vectors, use to sequence yeast selectable 
                     marker"
     primer_bind     12982..13004
                     /label=pGEX 3'
                     /note="pGEX vectors, reverse primer"
     primer_bind     complement(13042..13060)
                     /label=pBRforEco
                     /note="pBR322 vectors, upsteam of EcoRI site, forward 
                     primer"
     promoter        13128..13232
                     /gene="bla"
                     /label=AmpR promoter
     CDS             join(13233..13301,13302..14093)
                     /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(13451..13470)
                     /label=Amp-R
                     /note="Ampicillin resistance gene, reverse primer"
     rep_origin      14264..14852
                     /direction=RIGHT
                     /label=ori
                     /note="high-copy-number ColE1/pMB1/pBR322/pUC origin of 
                     replication"
     primer_bind     14753..14772
                     /label=pBR322ori-F
                     /note="pBR322 origin, forward primer"
     primer_bind     15006..15023
                     /label=L4440
                     /note="L4440 vector, forward primer"
ORIGIN
        1 tctccccgcg cgttggccga ttcattaatg cagctgtgga atgtgtgtca gttagggtgt
       61 ggaaagtccc caggctcccc agcaggcaga agtatgcaaa gcatgcatct caattagtca
      121 gcaaccaggt gtggaaagtc cccaggctcc ccagcaggca gaagtatgca aagcatgcat
      181 ctcaattagt cagcaaccat agtcccgccc ctaactccgc ccatcccgcc cctaactccg
      241 cccagttccg cccattctcc gccccatggc tgactaattt tttttattta tgcagaggcc
      301 gaggccgcct cggcctctga gctattccag aagtagtgag gaggcttttt tggaggccta
      361 ggcttttgca aaaagcttgg acacaagaca ggcttgcgag atatgtttga gaataccact
      421 ttatcccgcg tcagggagag gcagtgcgta aaaagacgcg gactcatgtg aaatactggt
      481 ttttagtgcg ccagatctct ataatctcgc gcaacctatt ttcccctcga acacttttta
      541 agccgtagat aaacaggctg ggacacttca catgagcgaa aaatacatcg tcacctggga
      601 catgttgcag atccatgcac gtaaactcgc aagccgactg atgccttctg aacaatggaa
      661 aggcattatt gccgtaagcc gtggcggtct gtaccgggtg cgttactggc gcgtgaactg
      721 ggtattcgtc atgtcgatac cgtttgtatt tccagctacg atcacgacaa ccagcgcgag
      781 cttaaagtgc tgaaacgcgc agaaggcgat ggcgaaggct tcatcgttat tgatgacctg
      841 gtggataccg gtggtactgc ggttgcgatt cgtgaaatgt atccaaaagc gcactttgtc
      901 accatcttcg caaaaccggc tggtcgtccg ctggttgatg actatgttgt tgatatcccg
      961 caagatacct ggattgaaca gccgtgggat atgggcgtcg tattcgtccc gccaatctcc
     1021 ggtcgctaat cttttcaacg cctggcactg ccgggcgttg ttctttttaa cttcaggcgg
     1081 gttacaatag tttccagtaa gtattctgga ggctgcatcc atgacacagg caaacctgag
     1141 cgaaaccctg ttcaaacccc gctttaaaca tcctgaaacc tcgacgctag tccgccgctt
     1201 taatcacggc gcacaaccgc ctgtgcagtc ggcccttgat ggtaaaacca tccctcactg
     1261 gtatcgcatg attaaccgtc tgatgtggat ctggcgcggc attgacccac gcgaaatcct
     1321 cgacgtccag gcacgtattg tgatgagcga tgccgaacgt accgacgatg atttatacga
     1381 tacggtgatt ggctaccgtg gcggcaactg gatttatgag tgggccccgg atctttgtga
     1441 aggaacctta cttctgtggt gtgacataat tggacaaact acctacagag atttaaagct
     1501 ctaaggtaaa tataaaattt ttaagtgtat aatgtgttaa actactgatt ctaattgttt
     1561 gtgtatttta gattccaacc tatggaactg atgaatggga gcagtggtgg aatgccttta
     1621 atgaggaaaa cctgttttgc tcagaagaaa tgccatctag tgatgatgag gctactgctg
     1681 actctcaaca ttctactcct ccaaaaaaga agagaaaggt agaagacccc aaggactttc
     1741 cttcagaatt gctaagtttt ttgagtcatg ctgtgtttag taatagaact cttgcttgct
     1801 ttgctattta caccacaaag gaaaaagctg cactgctata caagaaaatt atggaaaaat
     1861 attctgtaac ctttataagt aggcataaca gttataatca taacatactg ttttttctta
     1921 ctccacacag gcatagagtg tctgctatta ataactatgc tcaaaaattg tgtaccttta
     1981 gctttttaat ttgtaaaggg gttaataagg aatatttgat gtatagtgcc ttgactagag
     2041 atcataatca gccataccac atttgtagag gttttacttg ctttaaaaaa cctcccacac
     2101 ctccccctga acctgaaaca taaaatgaat gcaattgttg ttgttaactt gtttattgca
     2161 gcttataatg gttacaaata aagcaatagc atcacaaatt tcacaaataa agcatttttt
     2221 tcactgcatt ctagttgtgg tttgtccaaa ctcatcaatg tatcttatca tgtctggatc
     2281 aactggataa ctcaagctaa ccaaaatcat cccaaacttc ccaccccata ccctattacc
     2341 actgccaatt acctgtggtt tcatttactc taaacctgtg attcctctga attattttca
     2401 ttttaaagaa attgtatttg ttaaatatgt actacaaact tagtagttgg aagggctaat
     2461 tcactcccaa agaagacaag atatccttga tctgtggatc taccacacac aaggctactt
     2521 ccctgattag cagaactaca caccagggcc aggggtcaga tatccactga cctttggatg
     2581 gtgctacaag ctagtaccag ttgagccaga taaggtagaa gaggccaata aaggagagaa
     2641 caccagcttg ttacaccctg tgagcctgca tgggatggat gacccggaga gagaagtgtt
     2701 agagtggagg tttgacagcc gcctagcatt tcatcacgtg gcccgagagc tgcatccgga
     2761 gtacttcaag aactgctgat atcgagcttg ctacaaggga ctttccgctg gggactttcc
     2821 agggaggcgt ggcctgggcg ggactgggga gtggcgagcc ctcagatcct gcatataagc
     2881 agctgctttt tgcctgtact gggtctctct ggttagacca gatctgagcc tgggagctct
     2941 ctggctaact agggaaccca ctgcttaagc ctcaataaag cttgccttga gtgcttcaag
     3001 tagtgtgtgc ccgtctgttg tgtgactctg gtaactagag atccctcaga cccttttagt
     3061 cagtgtggaa aatctctagc agtggcgccc gaacagggac ttgaaagcga aagggaaacc
     3121 agaggagctc tctcgacgca ggactcggct tgctgaagcg cgcacggcaa gaggcgaggg
     3181 gcggcgactg gtgagtacgc caaaaatttt gactagcgga ggctagaagg agagagatgg
     3241 gtgcgagagc gtcagtatta agcgggggag aattagatcg cgatgggaaa aaattcggtt
     3301 aaggccaggg ggaaagaaaa aatataaatt aaaacatata gtatgggcaa gcagggagct
     3361 agaacgattc gcagttaatc ctggcctgtt agaaacatca gaaggctgta gacaaatact
     3421 gggacagcta caaccatccc ttcagacagg atcagaagaa cttagatcat tatataatac
     3481 agtagcaacc ctctattgtg tgcatcaaag gatagagata aaagacacca aggaagcttt
     3541 agacaagata gaggaagagc aaaacaaaag taagaccacc gcacagcaag cggccggccg
     3601 ctgatcttca gacctggagg aggagatatg agggacaatt ggagaagtga attatataaa
     3661 tataaagtag taaaaattga accattagga gtagcaccca ccaaggcaaa gagaagagtg
     3721 gtgcagagag aaaaaagagc agtgggaata ggagctttgt tccttgggtt cttgggagca
     3781 gcaggaagca ctatgggcgc agcgtcaatg acgctgacgg tacaggccag acaattattg
     3841 tctggtatag tgcagcagca gaacaatttg ctgagggcta ttgaggcgca acagcatctg
     3901 ttgcaactca cagtctgggg catcaagcag ctccaggcaa gaatcctggc tgtggaaaga
     3961 tacctaaagg atcaacagct cctggggatt tggggttgct ctggaaaact catttgcacc
     4021 actgctgtgc cttggaatgc tagttggagt aataaatctc tggaacagat ttggaatcac
     4081 acgacctgga tggagtggga cagagaaatt aacaattaca caagcttaat acactcctta
     4141 attgaagaat cgcaaaacca gcaagaaaag aatgaacaag aattattgga attagataaa
     4201 tgggcaagtt tgtggaattg gtttaacata acaaattggc tgtggtatat aaaattattc
     4261 ataatgatag taggaggctt ggtaggttta agaatagttt ttgctgtact ttctatagtg
     4321 aatagagtta ggcagggata ttcaccatta tcgtttcaga cccacctccc aaccccgagg
     4381 ggacccgaca ggcccgaagg aatagaagaa gaaggtggag agagagacag agacagatcc
     4441 attcgattag tgaacggatc tcgacggtat cgccaaatgg cagtattcat ccacaatttt
     4501 aaaagaaaag gggggattgg ggggtacagt gcaggggaaa gaatagtaga cataatagca
     4561 acagacatac aaactaaaga attacaaaaa caaattacaa aaattcaaaa ttttcgggtt
     4621 tattacaggg acagcagaga tccagtttgg atcgataagc ttgatatcga attcgggagg
     4681 tggtccctgc agttacgcca atgataaccc ccgccagaaa aatcttagta gccttccctt
     4741 tttgttttcc gtgccccaac tcggcggatt gactcggccc cttccggaaa cacccgaatc
     4801 aacttctagt caaattattg ttcacgccgc aatgacccac ccctggcccg cgtctgtgga
     4861 actgacccct ggtgtacagg agagttcgct gctgaaagtg gtcccaaagg ggtactagtt
     4921 tttaagctcc caactccccc tcccccagcg tctggaggat tccacaccct cgcaccgcag
     4981 gggcgaggaa gtgggcggag tccggttttg gcgccagccg ctgaggctgc caagcagaaa
     5041 agccaccgct gaggagactc cggtcactgt cctcgccccg cctccccctt ccctcccctt
     5101 ggggaccacc gggcgccacg ccgcgaacgg taagtgccgc ggtcgtcggc gcctccgccc
     5161 tccccctagg gccccaattc ccagcgggcg cggcgcgcgg ccccgcgcgc agctcccggc
     5221 tccctccccc ttcggatgtg gcttgagctg taggcgcgga gtcctgcagc cccgataaaa
     5281 taaaagattt tatttagtct ccagaaaaag gggggaatga aagaccccac ctgtaggttt
     5341 ggcaagctag ctgcagtaac gccattttgc aaggcatgga aaaataccaa accaagaata
     5401 gagaagttca gatcaagggc gggtacatga aaatagctaa cgttgggcca aacaggatat
     5461 ctgcggtgag cagtttcggc cccggcccgg ggccaagaac agatggtcac cgcagtttcg
     5521 gccccggccc gaggccaaga acagatggtc cccagatatg gcccaaccct cagcagtttc
     5581 ttaagaccca tcagatgttt ccaggctccc ccaaggacct gaaatgaccc tgcgccttat
     5641 ttgaattaac caatcagcct gcttctcgct tctgttcgcg cgcttctgct tcccgagctc
     5701 tataaaagag ctcacaaccc ctcactcggc gcgccagtcc tccgacagac tgagtcgccc
     5761 gggggggatc tggagctctc gagaattctc acgcgtctgc aggatatcaa gcttgcggta
     5821 ccgcgggccc ggccaccatg aacaattccc agggaagagt gaccttcgag gatgtcactg
     5881 tgaacttcac ccagggggag tggcagcggc tgaatcccga acagagaaac ttgtacaggg
     5941 atgtgatgct ggagaattac agcaaccttg tctctgtggg acaaggggaa accaccaaac
     6001 ccgatgtgat cttgaggttg gaacaaggaa aggagccatg gttggaggaa gaggaagtgc
     6061 tgggaagtgg ccgtgcagaa aaaaatgggg acattggagg gcagatttgg aagccaaagg
     6121 atgtgaaaga gagtctctac ccaactttct tgtacaaagt ggttggtgga tctggaggtt
     6181 ctcccaagaa gaagcgcaag gtgggacgcg tctgcaggat atcaagcttg cggtaccgcg
     6241 ggcccgggat cgccaccatg gacaagaagt acagcatcgg cctggccatc ggcaccaact
     6301 ctgtgggctg ggccgtgatc accgacgagt acaaggtgcc cagcaagaaa ttcaaggtgc
     6361 tgggcaacac cgaccggcac agcatcaaga agaacctgat cggcgccctg ctgttcgaca
     6421 gcggagaaac agccgaggcc acccggctga agagaaccgc cagaagaaga tacaccagac
     6481 ggaagaaccg gatctgctat ctgcaagaga tcttcagcaa cgagatggcc aaggtggacg
     6541 acagcttctt ccacagactg gaagagtcct tcctggtgga agaggataag aagcacgagc
     6601 ggcaccccat cttcggcaac atcgtggacg aggtggccta ccacgagaag taccccacca
     6661 tctaccacct gagaaagaaa ctggtggaca gcaccgacaa ggccgacctg cggctgatct
     6721 atctggccct ggcccacatg atcaagttcc ggggccactt cctgatcgag ggcgacctga
     6781 accccgacaa cagcgacgtg gacaagctgt tcatccagct ggtgcagacc tacaaccagc
     6841 tgttcgagga aaaccccatc aacgccagcg gcgtggacgc caaggccatc ctgtctgcca
     6901 gactgagcaa gagcagacgg ctggaaaatc tgatcgccca gctgcccggc gagaagaaga
     6961 atggcctgtt cggcaacctg attgccctga gcctgggcct gacccccaac ttcaagagca
     7021 acttcgacct ggccgaggat gccaaactgc agctgagcaa ggacacctac gacgacgacc
     7081 tggacaacct gctggcccag atcggcgacc agtacgccga cctgtttctg gccgccaaga
     7141 acctgtccga cgccatcctg ctgagcgaca tcctgagagt gaacaccgag atcaccaagg
     7201 cccccctgag cgcctctatg atcaagagat acgacgagca ccaccaggac ctgaccctgc
     7261 tgaaagctct cgtgcggcag cagctgcctg agaagtacaa agagattttc ttcgaccaga
     7321 gcaagaacgg ctacgccggc tacatcgatg gcggagccag ccaggaagag ttctacaagt
     7381 tcatcaagcc catcctggaa aagatggacg gcaccgagga actgctcgtg aagctgaaca
     7441 gagaggacct gctgcggaag cagcggacct tcgacaacgg cagcatcccc caccagatcc
     7501 acctgggaga gctgcacgcc attctgcggc ggcaggaaga tttttaccca ttcctgaagg
     7561 acaaccggga aaagatcgag aagatcctga ccttccgcat cccctactac gtgggccctc
     7621 tggccagggg aaacagcaga ttcgcctgga tgaccagaaa gagcgaggaa accatcaccc
     7681 cctggaactt cgaggaagtg gtggacaagg gcgccagcgc ccagagcttc atcgagcgga
     7741 tgaccaactt cgataagaac ctgcccaacg agaaggtgct gcccaagcac agcctgctgt
     7801 acgagtactt caccgtgtac aacgagctga ccaaagtgaa atacgtgacc gagggaatga
     7861 gaaagcccgc cttcctgagc ggcgagcaga aaaaagccat cgtggacctg ctgttcaaga
     7921 ccaaccggaa agtgaccgtg aagcagctga aagaggacta cttcaagaaa atcgagtgct
     7981 tcgactccgt ggaaatctcc ggcgtggaag atcggttcaa cgcctccctg ggcacatacc
     8041 acgatctgct gaaaattatc aaggacaagg acttcctgga caatgaggaa aacgaggaca
     8101 ttctggaaga tatcgtgctg accctgacac tgtttgagga cagagagatg atcgaggaac
     8161 ggctgaaaac ctatgcccac ctgttcgacg acaaagtgat gaagcagctg aagcggcgga
     8221 gatacaccgg ctggggcagg ctgagccgga agctgatcaa cggcatccgg gacaagcagt
     8281 ccggcaagac aatcctggat ttcctgaagt ccgacggctt cgccaacaga aacttcatgc
     8341 agctgatcca cgacgacagc ctgaccttta aagaggacat ccagaaagcc caggtgtccg
     8401 gccagggcga tagcctgcac gagcacattg ccaatctggc cggcagcccc gccattaaga
     8461 agggcatcct gcagacagtg aaggtggtgg acgagctcgt gaaagtgatg ggccggcaca
     8521 agcccgagaa catcgtgatc gaaatggcca gagagaacca gaccacccag aagggacaga
     8581 agaacagccg cgagagaatg aagcggatcg aagagggcat caaagagctg ggcagccaga
     8641 tcctgaaaga acaccccgtg gaaaacaccc agctgcagaa cgagaagctg tacctgtact
     8701 acctgcagaa tgggcgggat atgtacgtgg accaggaact ggacatcaac cggctgtccg
     8761 actacgatgt ggacgctatc gtgcctcaga gctttctgaa ggacgactcc atcgataaca
     8821 aagtgctgac tcggagcgac aagaaccggg gcaagagcga caacgtgccc tccgaagagg
     8881 tcgtgaagaa gatgaagaac tactggcgcc agctgctgaa tgccaagctg attacccaga
     8941 ggaagttcga caatctgacc aaggccgaga gaggcggcct gagcgaactg gataaggccg
     9001 gcttcatcaa gagacagctg gtggaaaccc ggcagatcac aaagcacgtg gcacagatcc
     9061 tggactcccg gatgaacact aagtacgacg agaacgacaa actgatccgg gaagtgaaag
     9121 tgatcaccct gaagtccaag ctggtgtccg atttccggaa ggatttccag ttttacaaag
     9181 tgcgcgagat caacaactac caccacgccc acgacgccta cctgaacgcc gtcgtgggaa
     9241 ccgccctgat caaaaagtac cctaagctgg aaagcgagtt cgtgtacggc gactacaagg
     9301 tgtacgacgt gcggaagatg atcgccaaga gcgagcagga aatcggcaag gctaccgcca
     9361 agtacttctt ctacagcaac atcatgaact ttttcaagac cgagattacc ctggccaacg
     9421 gcgagatccg gaagcggcct ctgatcgaga caaacggcga aacaggcgag atcgtgtggg
     9481 ataagggccg ggactttgcc accgtgcgga aagtgctgtc tatgccccaa gtgaatatcg
     9541 tgaaaaagac cgaggtgcag acaggcggct tcagcaaaga gtctatcctg cccaagagga
     9601 acagcgacaa gctgatcgcc agaaagaagg actgggaccc taagaagtac ggcggcttcg
     9661 acagccccac cgtggcctat tctgtgctgg tggtggccaa agtggaaaag ggcaagtcca
     9721 agaaactgaa gagtgtgaaa gagctgctgg ggatcaccat catggaaaga agcagcttcg
     9781 agaagaatcc catcgacttt ctggaagcca agggctacaa agaagtgaaa aaggacctga
     9841 tcatcaagct gcctaagtac tccctgttcg agctggaaaa cggccggaag agaatgctgg
     9901 cctctgccgg cgaactgcag aagggaaacg aactggccct gccctccaaa tatgtgaact
     9961 tcctgtacct ggccagccac tatgagaagc tgaagggctc ccccgaggat aatgagcaga
    10021 aacagctgtt tgtggaacag cacaaacact acctggacga gatcatcgag cagatcagcg
    10081 agttctccaa gagagtgatc ctggccgacg ctaatctgga caaggtgctg agcgcctaca
    10141 acaagcacag agacaagcct atcagagagc aggccgagaa tatcatccac ctgtttaccc
    10201 tgaccaatct gggagcccct gccgccttca agtactttga caccaccatc gaccggaaga
    10261 ggtacaccag caccaaagag gtgctggacg ccaccctgat ccaccagagc atcaccggcc
    10321 tgtacgagac acggatcgac ctgtctcagc tgggaggcga cgcctatccc tatgacgtgc
    10381 ccgattatgc cagcctgggc agcggctccc ccaagaaaaa acgcaaggtg gaagatccta
    10441 agaaaaagcg gaaagtgagc ggccgcataa cttcgtataa agtatcctat acgaagttat
    10501 ccgctactaa cttcagcctg ctgaagcagg ctggagacgt ggaggagaac cctggacctg
    10561 tgagcaaggg cgaggagctg ttcaccgggg tggtgcccat cctggtcgag ctggacggcg
    10621 acgtaaacgg ccacaagttc agcgtgtctg gcgagggcga gggcgatgcc acctacggca
    10681 agctgaccct gaagttcatc tgcaccaccg gcaagctgcc cgtgccctgg cccaccctcg
    10741 tgaccaccct gacctacggc gtgcagtgct tcagccgcta ccccgaccac atgaagcagc
    10801 acgacttctt caagtccgcc atgcccgaag gctacgtcca ggagcgcacc atcttcttca
    10861 aggacgacgg caactacaag acccgcgccg aggtgaagtt cgagggcgac accctggtga
    10921 accgcatcga gctgaagggc atcgacttca aggaggacgg caacatcctg gggcacaagc
    10981 tggagtacaa ctacaacagc cacaacgtct atatcatggc cgacaagcag aagaacggca
    11041 tcaaggcgaa cttcaagatc cgccacaaca tcgaggacgg cagcgtgcag ctcgccgacc
    11101 actaccagca gaacaccccc atcggcgacg gccccgtgct gctgcccgac aaccactacc
    11161 tgagcaccca gtccgccctg agcaaagacc ccaacgagaa gcgcgatcac atggtcctgc
    11221 tggagttcgt gaccgccgcc gggatcactc tcggcatgga cgagctgtac aagtaataac
    11281 ttcgtataaa gtatcctata cgaagttatc caagtagtga gcgatcgccc tgcaggcatg
    11341 caagcttgat atcaagctta tcgataatca acctctggat tacaaaattt gtgaaagatt
    11401 gactggtatt cttaactatg ttgctccttt tacgctatgt ggatacgctg ctttaatgcc
    11461 tttgtatcat gctattgctt cccgtatggc tttcattttc tcctccttgt ataaatcctg
    11521 gttgctgtct ctttatgagg agttgtggcc cgttgtcagg caacgtggcg tggtgtgcac
    11581 tgtgtttgct gacgcaaccc ccactggttg gggcattgcc accacctgtc agctcctttc
    11641 cgggactttc gctttccccc tccctattgc cacggcggaa ctcatcgccg cctgccttgc
    11701 ccgctgctgg acaggggctc ggctgttggg cactgacaat tccgtggtgt tgtcggggaa
    11761 atcatcgtcc tttccttggc tgctcgcctg tgttgccacc tggattctgc gcgggacgtc
    11821 cttctgctac gtcccttcgg ccctcaatcc agcggacctt ccttcccgcg gcctgctgcc
    11881 ggctctgcgg cctcttccgc gtcttcgcct tcgccctcag acgagtcgga tctccctttg
    11941 ggccgcctcc ccgcatcgat accgtcgacc tcgagggaat taattcgagc tcggtacctt
    12001 taagaccaat gacttacaag gcagctgtag atcttagcca ctttttaaaa gaaaaggggg
    12061 gactggaagg gctaattcac tcccaacgaa gacaagatct gctttttgct tgtactgggt
    12121 ctctctggtt agaccagatc tgagcctggg agctctctgg ctaactaggg aacccactgc
    12181 ttaagcctca ataaagcttg ccttgagtgc ttcaagtagt gtgtgcccgt ctgttgtgtg
    12241 actctggtaa ctagagatcc ctcagaccct tttagtcagt gtggaaaatc tctagcagca
    12301 tctagaatta attccgtgta ttctatagtg tcacctaaat cgtatgtgta tgatacataa
    12361 ggttatgtat taattgtagc cgcgttctaa cgacaatatg tacaagccta attgtgtagc
    12421 atctggctta ctgaagcaga ccctatcatc tctctcgtaa actgccgtca gagtcggttt
    12481 ggttggacga accttctgag tttctggtaa cgccgtcccg cacccggaaa tggtcagcga
    12541 accaatcagc agggtcatcg ctagccagat cctctacgcc ggacgcatcg tggccggcat
    12601 caccggcgcc acaggtgcgg ttgctggcgc ctatatcgcc gacatcaccg atggggaaga
    12661 tcgggctcgc cacttcgggc tcatgagcgc ttgtttcggc gtgggtatgg tggcaggccc
    12721 cgtggccggg ggactgttgg gcgccatctc cttgcatgca ccattccttg cggcggcggt
    12781 gctcaacggc ctcaacctac tactgggctg cttcctaatg caggagtcgc ataagggaga
    12841 gcgtcgaatg gtgcactctc agtacaatct gctctgatgc cgcatagtta agccagcccc
    12901 gacacccgcc aacacccgct gacgcgccct gacgggcttg tctgctcccg gcatccgctt
    12961 acagacaagc tgtgaccgtc tccgggagct gcatgtgtca gaggttttca ccgtcatcac
    13021 cgaaacgcgc gagacgaaag ggcctcgtga tacgcctatt tttataggtt aatgtcatga
    13081 taataatggt ttcttagacg tcaggtggca cttttcgggg aaatgtgcgc ggaaccccta
    13141 tttgtttatt tttctaaata cattcaaata tgtatccgct catgagacaa taaccctgat
    13201 aaatgcttca ataatattga aaaaggaaga gtatgagtat tcaacatttc cgtgtcgccc
    13261 ttattccctt ttttgcggca ttttgccttc ctgtttttgc tcacccagaa acgctggtga
    13321 aagtaaaaga tgctgaagat cagttgggtg cacgagtggg ttacatcgaa ctggatctca
    13381 acagcggtaa gatccttgag agttttcgcc ccgaagaacg ttttccaatg atgagcactt
    13441 ttaaagttct gctatgtggc gcggtattat cccgtattga cgccgggcaa gagcaactcg
    13501 gtcgccgcat acactattct cagaatgact tggttgagta ctcaccagtc acagaaaagc
    13561 atcttacgga tggcatgaca gtaagagaat tatgcagtgc tgccataacc atgagtgata
    13621 acactgcggc caacttactt ctgacaacga tcggaggacc gaaggagcta accgcttttt
    13681 tgcacaacat gggggatcat gtaactcgcc ttgatcgttg ggaaccggag ctgaatgaag
    13741 ccataccaaa cgacgagcgt gacaccacga tgcctgtagc aatggcaaca acgttgcgca
    13801 aactattaac tggcgaacta cttactctag cttcccggca acaattaata gactggatgg
    13861 aggcggataa agttgcagga ccacttctgc gctcggccct tccggctggc tggtttattg
    13921 ctgataaatc tggagccggt gagcgtgggt ctcgcggtat cattgcagca ctggggccag
    13981 atggtaagcc ctcccgtatc gtagttatct acacgacggg gagtcaggca actatggatg
    14041 aacgaaatag acagatcgct gagataggtg cctcactgat taagcattgg taactgtcag
    14101 accaagttta ctcatatata ctttagattg atttaaaact tcatttttaa tttaaaagga
    14161 tctaggtgaa gatccttttt gataatctca tgaccaaaat cccttaacgt gagttttcgt
    14221 tccactgagc gtcagacccc gtagaaaaga tcaaaggatc ttcttgagat cctttttttc
    14281 tgcgcgtaat ctgctgcttg caaacaaaaa aaccaccgct accagcggtg gtttgtttgc
    14341 cggatcaaga gctaccaact ctttttccga aggtaactgg cttcagcaga gcgcagatac
    14401 caaatactgt tcttctagtg tagccgtagt taggccacca cttcaagaac tctgtagcac
    14461 cgcctacata cctcgctctg ctaatcctgt taccagtggc tgctgccagt ggcgataagt
    14521 cgtgtcttac cgggttggac tcaagacgat agttaccgga taaggcgcag cggtcgggct
    14581 gaacgggggg ttcgtgcaca cagcccagct tggagcgaac gacctacacc gaactgagat
    14641 acctacagcg tgagctatga gaaagcgcca cgcttcccga agggagaaag gcggacaggt
    14701 atccggtaag cggcagggtc ggaacaggag agcgcacgag ggagcttcca gggggaaacg
    14761 cctggtatct ttatagtcct gtcgggtttc gccacctctg acttgagcgt cgatttttgt
    14821 gatgctcgtc aggggggcgg agcctatgga aaaacgccag caacgcggcc tttttacggt
    14881 tcctggcctt ttgctggcct tttgctcaca tgttctttcc tgcgttatcc cctgattctg
    14941 tggataaccg tattaccgcc tttgagtgag ctgataccgc tcgccgcagc cgaacgaccg
    15001 agcgcagcga gtcagtgagc gaggaagcgg aagagcgccc aatacgcaaa ccgcc
//
