LOCUS       sequence-374880-       15284 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 15284)
  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 15284)
  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-P2A-Hygro
FEATURES             Location/Qualifiers
     source          1..15284
                     /mol_type="other DNA"
                     /organism="synthetic DNA construct"
     promoter        complement(47..65)
                     /label=SP6 promoter
                     /note="promoter for bacteriophage SP6 RNA polymerase"
     primer_bind     complement(48..65)
                     /label=SP6
                     /note="SP6 promoter, forward primer"
     primer_bind     complement(355..374)
                     /label=pBRrevBam
                     /note="pBR322 vectors, tet region, downstream of BamHI, 
                     reverse primer"
     primer_bind     complement(588..607)
                     /label=pRS-marker
                     /note="pRS vectors, use to sequence yeast selectable 
                     marker"
     primer_bind     707..729
                     /label=pGEX 3'
                     /note="pGEX vectors, reverse primer"
     primer_bind     complement(767..785)
                     /label=pBRforEco
                     /note="pBR322 vectors, upsteam of EcoRI site, forward 
                     primer"
     promoter        853..957
                     /gene="bla"
                     /label=AmpR promoter
     CDS             join(958..1026,1027..1818)
                     /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(1176..1195)
                     /label=Amp-R
                     /note="Ampicillin resistance gene, reverse primer"
     rep_origin      1989..2577
                     /direction=RIGHT
                     /label=ori
                     /note="high-copy-number ColE1/pMB1/pBR322/pUC origin of 
                     replication"
     primer_bind     2478..2497
                     /label=pBR322ori-F
                     /note="pBR322 origin, forward primer"
     primer_bind     2731..2748
                     /label=L4440
                     /note="L4440 vector, forward primer"
     primer_bind     complement(2818..2838)
                     /label=pBABE 3'
                     /note="SV40 enhancer, reverse primer for pBABE vectors"
     promoter        2823..3152
                     /label=SV40 promoter
                     /note="SV40 enhancer and early promoter"
     rep_origin      3003..3138
                     /label=SV40 ori
                     /note="SV40 origin of replication"
     intron          4286..4351
                     /label=small t intron
                     /note="SV40 (simian virus 40) small t antigen intron"
     CDS             4481..4501
                     /codon_start=1
                     /product="nuclear localization signal of SV40 (simian virus
                     40) large T antigen"
                     /label=SV40 NLS
                     /translation="PKKKRKV"
     polyA_signal    4926..5060
                     /label=SV40 poly(A) signal
                     /note="SV40 polyadenylation signal"
     primer_bind     complement(4963..4982)
                     /label=SV40pA-R
                     /note="SV40 polyA, reverse primer"
     primer_bind     5017..5036
                     /label=EBV-rev
                     /note="SV40 polyA terminator, reverse primer"
     LTR             5228..5861
                     /label=3' LTR
                     /note="3' long terminal repeat (LTR) from HIV-1"
     misc_feature    5908..6033
                     /label=HIV-1 Psi
                     /note="packaging signal of human immunodeficiency virus 
                     type 1"
     misc_feature    6530..6763
                     /label=RRE
                     /note="The Rev response element (RRE) of HIV-1 allows for 
                     Rev-dependent mRNA export from the nucleus to the 
                     cytoplasm."
     CDS             6948..6992
                     /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    7277..7394
                     /label=cPPT/CTS
                     /note="central polypurine tract and central termination 
                     sequence of HIV-1"
     promoter        8136..8543
                     /label=SFFV promoter
                     /note="spleen focus-forming virus long terminal repeat 
                     (LTR) promoter"
     protein_bind    complement(8919..8943)
                     /gene="mutant version of attB"
                     /label=attB2
                     /bound_moiety="BP Clonase(TM)"
                     /note="recombination site for the Gateway(R) BP reaction"
     CDS             8963..8983
                     /codon_start=1
                     /product="nuclear localization signal of SV40 (simian virus
                     40) large T antigen"
                     /label=SV40 NLS
                     /translation="PKKKRKV"
     regulatory      9032..9041
                     /label=Kozak sequence
                     /note="vertebrate consensus sequence for strong initiation 
                     of translation (Kozak, 1987)"
                     /regulatory_class="other"
     CDS             join(9038..9064,9065..9067,9068..11554,11555..11557,
                     11558..13141)
                     /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             9038..13141
                     /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             13145..13171
                     /codon_start=1
                     /product="HA (human influenza hemagglutinin) epitope tag"
                     /label=HA
                     /translation="YPYDVPDYA"
     CDS             13151..13177
                     /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             13190..13210
                     /codon_start=1
                     /product="nuclear localization signal of SV40 (simian virus
                     40) large T antigen"
                     /label=SV40 NLS
                     /translation="PKKKRKV"
     CDS             13217..13237
                     /codon_start=1
                     /product="nuclear localization signal of SV40 (simian virus
                     40) large T antigen"
                     /label=SV40 NLS
                     /translation="PKKKRKV"
     CDS             13247..13303
                     /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             13304..14326
                     /codon_start=1
                     /product="hygromycin B phosphotransferase"
                     /label=HygR
                     /note="confers resistance to hygromycin"
                     /translation="KKPELTATSVEKFLIEKFDSVSDLMQLSEGEESRAFSFDVGGRGY
                     VLRVNSCADGFYKDRYVYRHFASAALPIPEVLDIGEFSESLTYCISRRAQGVTLQDLPE
                     TELPAVLQPVAEAMDAIAAADLSQTSGFGPFGPQGIGQYTTWRDFICAIADPHVYHWQT
                     VMDDTVSASVAQALDELMLWAEDCPEVRHLVHADFGSNNVLTDNGRITAVIDWSEAMFG
                     DSQYEVANIFFWRPWLACMEQQTRYFERRHPELAGSPRLRAYMLRIGLDQLYQSLVDGN
                     FDDAAWAQGRCDAIVRSGAGTVGRTQIARRSAAVWTDGCVEVLADSGNRRPSTRPRAKE
                     "
     misc_feature    14375..14963
                     /label=WPRE
                     /note="woodchuck hepatitis virus posttranscriptional 
                     regulatory element"
     primer_bind     complement(14428..14448)
                     /label=WPRE-R
                     /note="WPRE, reverse primer"
     CDS             complement(14846..14857)
                     /codon_start=1
                     /product="Factor Xa recognition and cleavage site"
                     /label=Factor Xa site
                     /translation="IEGR"
     primer_bind     complement(14966..14982)
                     /label=KS primer
                     /note="common sequencing primer, one of multiple similar 
                     variants"
     primer_bind     complement(14967..14983)
                     /label=pBluescriptKS
                     /note="For pBluescript vector"
     LTR             join(15073..15284,1..22)
                     /label=3' LTR (Delta-U3)
                     /note="self-inactivating 3' long terminal repeat (LTR) from
                     HIV-1"
ORIGIN
        1 tcagtgtgga aaatctctag cagcatctag aattaattcc gtgtattcta tagtgtcacc
       61 taaatcgtat gtgtatgata cataaggtta tgtattaatt gtagccgcgt tctaacgaca
      121 atatgtacaa gcctaattgt gtagcatctg gcttactgaa gcagacccta tcatctctct
      181 cgtaaactgc cgtcagagtc ggtttggttg gacgaacctt ctgagtttct ggtaacgccg
      241 tcccgcaccc ggaaatggtc agcgaaccaa tcagcagggt catcgctagc cagatcctct
      301 acgccggacg catcgtggcc ggcatcaccg gcgccacagg tgcggttgct ggcgcctata
      361 tcgccgacat caccgatggg gaagatcggg ctcgccactt cgggctcatg agcgcttgtt
      421 tcggcgtggg tatggtggca ggccccgtgg ccgggggact gttgggcgcc atctccttgc
      481 atgcaccatt ccttgcggcg gcggtgctca acggcctcaa cctactactg ggctgcttcc
      541 taatgcagga gtcgcataag ggagagcgtc gaatggtgca ctctcagtac aatctgctct
      601 gatgccgcat agttaagcca gccccgacac ccgccaacac ccgctgacgc gccctgacgg
      661 gcttgtctgc tcccggcatc cgcttacaga caagctgtga ccgtctccgg gagctgcatg
      721 tgtcagaggt tttcaccgtc atcaccgaaa cgcgcgagac gaaagggcct cgtgatacgc
      781 ctatttttat aggttaatgt catgataata atggtttctt agacgtcagg tggcactttt
      841 cggggaaatg tgcgcggaac ccctatttgt ttatttttct aaatacattc aaatatgtat
      901 ccgctcatga gacaataacc ctgataaatg cttcaataat attgaaaaag gaagagtatg
      961 agtattcaac atttccgtgt cgcccttatt cccttttttg cggcattttg ccttcctgtt
     1021 tttgctcacc cagaaacgct ggtgaaagta aaagatgctg aagatcagtt gggtgcacga
     1081 gtgggttaca tcgaactgga tctcaacagc ggtaagatcc ttgagagttt tcgccccgaa
     1141 gaacgttttc caatgatgag cacttttaaa gttctgctat gtggcgcggt attatcccgt
     1201 attgacgccg ggcaagagca actcggtcgc cgcatacact attctcagaa tgacttggtt
     1261 gagtactcac cagtcacaga aaagcatctt acggatggca tgacagtaag agaattatgc
     1321 agtgctgcca taaccatgag tgataacact gcggccaact tacttctgac aacgatcgga
     1381 ggaccgaagg agctaaccgc ttttttgcac aacatggggg atcatgtaac tcgccttgat
     1441 cgttgggaac cggagctgaa tgaagccata ccaaacgacg agcgtgacac cacgatgcct
     1501 gtagcaatgg caacaacgtt gcgcaaacta ttaactggcg aactacttac tctagcttcc
     1561 cggcaacaat taatagactg gatggaggcg gataaagttg caggaccact tctgcgctcg
     1621 gcccttccgg ctggctggtt tattgctgat aaatctggag ccggtgagcg tgggtctcgc
     1681 ggtatcattg cagcactggg gccagatggt aagccctccc gtatcgtagt tatctacacg
     1741 acggggagtc aggcaactat ggatgaacga aatagacaga tcgctgagat aggtgcctca
     1801 ctgattaagc attggtaact gtcagaccaa gtttactcat atatacttta gattgattta
     1861 aaacttcatt tttaatttaa aaggatctag gtgaagatcc tttttgataa tctcatgacc
     1921 aaaatccctt aacgtgagtt ttcgttccac tgagcgtcag accccgtaga aaagatcaaa
     1981 ggatcttctt gagatccttt ttttctgcgc gtaatctgct gcttgcaaac aaaaaaacca
     2041 ccgctaccag cggtggtttg tttgccggat caagagctac caactctttt tccgaaggta
     2101 actggcttca gcagagcgca gataccaaat actgttcttc tagtgtagcc gtagttaggc
     2161 caccacttca agaactctgt agcaccgcct acatacctcg ctctgctaat cctgttacca
     2221 gtggctgctg ccagtggcga taagtcgtgt cttaccgggt tggactcaag acgatagtta
     2281 ccggataagg cgcagcggtc gggctgaacg gggggttcgt gcacacagcc cagcttggag
     2341 cgaacgacct acaccgaact gagataccta cagcgtgagc tatgagaaag cgccacgctt
     2401 cccgaaggga gaaaggcgga caggtatccg gtaagcggca gggtcggaac aggagagcgc
     2461 acgagggagc ttccaggggg aaacgcctgg tatctttata gtcctgtcgg gtttcgccac
     2521 ctctgacttg agcgtcgatt tttgtgatgc tcgtcagggg ggcggagcct atggaaaaac
     2581 gccagcaacg cggccttttt acggttcctg gccttttgct ggccttttgc tcacatgttc
     2641 tttcctgcgt tatcccctga ttctgtggat aaccgtatta ccgcctttga gtgagctgat
     2701 accgctcgcc gcagccgaac gaccgagcgc agcgagtcag tgagcgagga agcggaagag
     2761 cgcccaatac gcaaaccgcc tctccccgcg cgttggccga ttcattaatg cagctgtgga
     2821 atgtgtgtca gttagggtgt ggaaagtccc caggctcccc agcaggcaga agtatgcaaa
     2881 gcatgcatct caattagtca gcaaccaggt gtggaaagtc cccaggctcc ccagcaggca
     2941 gaagtatgca aagcatgcat ctcaattagt cagcaaccat agtcccgccc ctaactccgc
     3001 ccatcccgcc cctaactccg cccagttccg cccattctcc gccccatggc tgactaattt
     3061 tttttattta tgcagaggcc gaggccgcct cggcctctga gctattccag aagtagtgag
     3121 gaggcttttt tggaggccta ggcttttgca aaaagcttgg acacaagaca ggcttgcgag
     3181 atatgtttga gaataccact ttatcccgcg tcagggagag gcagtgcgta aaaagacgcg
     3241 gactcatgtg aaatactggt ttttagtgcg ccagatctct ataatctcgc gcaacctatt
     3301 ttcccctcga acacttttta agccgtagat aaacaggctg ggacacttca catgagcgaa
     3361 aaatacatcg tcacctggga catgttgcag atccatgcac gtaaactcgc aagccgactg
     3421 atgccttctg aacaatggaa aggcattatt gccgtaagcc gtggcggtct gtaccgggtg
     3481 cgttactggc gcgtgaactg ggtattcgtc atgtcgatac cgtttgtatt tccagctacg
     3541 atcacgacaa ccagcgcgag cttaaagtgc tgaaacgcgc agaaggcgat ggcgaaggct
     3601 tcatcgttat tgatgacctg gtggataccg gtggtactgc ggttgcgatt cgtgaaatgt
     3661 atccaaaagc gcactttgtc accatcttcg caaaaccggc tggtcgtccg ctggttgatg
     3721 actatgttgt tgatatcccg caagatacct ggattgaaca gccgtgggat atgggcgtcg
     3781 tattcgtccc gccaatctcc ggtcgctaat cttttcaacg cctggcactg ccgggcgttg
     3841 ttctttttaa cttcaggcgg gttacaatag tttccagtaa gtattctgga ggctgcatcc
     3901 atgacacagg caaacctgag cgaaaccctg ttcaaacccc gctttaaaca tcctgaaacc
     3961 tcgacgctag tccgccgctt taatcacggc gcacaaccgc ctgtgcagtc ggcccttgat
     4021 ggtaaaacca tccctcactg gtatcgcatg attaaccgtc tgatgtggat ctggcgcggc
     4081 attgacccac gcgaaatcct cgacgtccag gcacgtattg tgatgagcga tgccgaacgt
     4141 accgacgatg atttatacga tacggtgatt ggctaccgtg gcggcaactg gatttatgag
     4201 tgggccccgg atctttgtga aggaacctta cttctgtggt gtgacataat tggacaaact
     4261 acctacagag atttaaagct ctaaggtaaa tataaaattt ttaagtgtat aatgtgttaa
     4321 actactgatt ctaattgttt gtgtatttta gattccaacc tatggaactg atgaatggga
     4381 gcagtggtgg aatgccttta atgaggaaaa cctgttttgc tcagaagaaa tgccatctag
     4441 tgatgatgag gctactgctg actctcaaca ttctactcct ccaaaaaaga agagaaaggt
     4501 agaagacccc aaggactttc cttcagaatt gctaagtttt ttgagtcatg ctgtgtttag
     4561 taatagaact cttgcttgct ttgctattta caccacaaag gaaaaagctg cactgctata
     4621 caagaaaatt atggaaaaat attctgtaac ctttataagt aggcataaca gttataatca
     4681 taacatactg ttttttctta ctccacacag gcatagagtg tctgctatta ataactatgc
     4741 tcaaaaattg tgtaccttta gctttttaat ttgtaaaggg gttaataagg aatatttgat
     4801 gtatagtgcc ttgactagag atcataatca gccataccac atttgtagag gttttacttg
     4861 ctttaaaaaa cctcccacac ctccccctga acctgaaaca taaaatgaat gcaattgttg
     4921 ttgttaactt gtttattgca gcttataatg gttacaaata aagcaatagc atcacaaatt
     4981 tcacaaataa agcatttttt tcactgcatt ctagttgtgg tttgtccaaa ctcatcaatg
     5041 tatcttatca tgtctggatc aactggataa ctcaagctaa ccaaaatcat cccaaacttc
     5101 ccaccccata ccctattacc actgccaatt acctgtggtt tcatttactc taaacctgtg
     5161 attcctctga attattttca ttttaaagaa attgtatttg ttaaatatgt actacaaact
     5221 tagtagttgg aagggctaat tcactcccaa agaagacaag atatccttga tctgtggatc
     5281 taccacacac aaggctactt ccctgattag cagaactaca caccagggcc aggggtcaga
     5341 tatccactga cctttggatg gtgctacaag ctagtaccag ttgagccaga taaggtagaa
     5401 gaggccaata aaggagagaa caccagcttg ttacaccctg tgagcctgca tgggatggat
     5461 gacccggaga gagaagtgtt agagtggagg tttgacagcc gcctagcatt tcatcacgtg
     5521 gcccgagagc tgcatccgga gtacttcaag aactgctgat atcgagcttg ctacaaggga
     5581 ctttccgctg gggactttcc agggaggcgt ggcctgggcg ggactgggga gtggcgagcc
     5641 ctcagatcct gcatataagc agctgctttt tgcctgtact gggtctctct ggttagacca
     5701 gatctgagcc tgggagctct ctggctaact agggaaccca ctgcttaagc ctcaataaag
     5761 cttgccttga gtgcttcaag tagtgtgtgc ccgtctgttg tgtgactctg gtaactagag
     5821 atccctcaga cccttttagt cagtgtggaa aatctctagc agtggcgccc gaacagggac
     5881 ttgaaagcga aagggaaacc agaggagctc tctcgacgca ggactcggct tgctgaagcg
     5941 cgcacggcaa gaggcgaggg gcggcgactg gtgagtacgc caaaaatttt gactagcgga
     6001 ggctagaagg agagagatgg gtgcgagagc gtcagtatta agcgggggag aattagatcg
     6061 cgatgggaaa aaattcggtt aaggccaggg ggaaagaaaa aatataaatt aaaacatata
     6121 gtatgggcaa gcagggagct agaacgattc gcagttaatc ctggcctgtt agaaacatca
     6181 gaaggctgta gacaaatact gggacagcta caaccatccc ttcagacagg atcagaagaa
     6241 cttagatcat tatataatac agtagcaacc ctctattgtg tgcatcaaag gatagagata
     6301 aaagacacca aggaagcttt agacaagata gaggaagagc aaaacaaaag taagaccacc
     6361 gcacagcaag cggccggccg ctgatcttca gacctggagg aggagatatg agggacaatt
     6421 ggagaagtga attatataaa tataaagtag taaaaattga accattagga gtagcaccca
     6481 ccaaggcaaa gagaagagtg gtgcagagag aaaaaagagc agtgggaata ggagctttgt
     6541 tccttgggtt cttgggagca gcaggaagca ctatgggcgc agcgtcaatg acgctgacgg
     6601 tacaggccag acaattattg tctggtatag tgcagcagca gaacaatttg ctgagggcta
     6661 ttgaggcgca acagcatctg ttgcaactca cagtctgggg catcaagcag ctccaggcaa
     6721 gaatcctggc tgtggaaaga tacctaaagg atcaacagct cctggggatt tggggttgct
     6781 ctggaaaact catttgcacc actgctgtgc cttggaatgc tagttggagt aataaatctc
     6841 tggaacagat ttggaatcac acgacctgga tggagtggga cagagaaatt aacaattaca
     6901 caagcttaat acactcctta attgaagaat cgcaaaacca gcaagaaaag aatgaacaag
     6961 aattattgga attagataaa tgggcaagtt tgtggaattg gtttaacata acaaattggc
     7021 tgtggtatat aaaattattc ataatgatag taggaggctt ggtaggttta agaatagttt
     7081 ttgctgtact ttctatagtg aatagagtta ggcagggata ttcaccatta tcgtttcaga
     7141 cccacctccc aaccccgagg ggacccgaca ggcccgaagg aatagaagaa gaaggtggag
     7201 agagagacag agacagatcc attcgattag tgaacggatc tcgacggtat cgccaaatgg
     7261 cagtattcat ccacaatttt aaaagaaaag gggggattgg ggggtacagt gcaggggaaa
     7321 gaatagtaga cataatagca acagacatac aaactaaaga attacaaaaa caaattacaa
     7381 aaattcaaaa ttttcgggtt tattacaggg acagcagaga tccagtttgg atcgataagc
     7441 ttgatatcga attcgggagg tggtccctgc agttacgcca atgataaccc ccgccagaaa
     7501 aatcttagta gccttccctt tttgttttcc gtgccccaac tcggcggatt gactcggccc
     7561 cttccggaaa cacccgaatc aacttctagt caaattattg ttcacgccgc aatgacccac
     7621 ccctggcccg cgtctgtgga actgacccct ggtgtacagg agagttcgct gctgaaagtg
     7681 gtcccaaagg ggtactagtt tttaagctcc caactccccc tcccccagcg tctggaggat
     7741 tccacaccct cgcaccgcag gggcgaggaa gtgggcggag tccggttttg gcgccagccg
     7801 ctgaggctgc caagcagaaa agccaccgct gaggagactc cggtcactgt cctcgccccg
     7861 cctccccctt ccctcccctt ggggaccacc gggcgccacg ccgcgaacgg taagtgccgc
     7921 ggtcgtcggc gcctccgccc tccccctagg gccccaattc ccagcgggcg cggcgcgcgg
     7981 ccccgcgcgc agctcccggc tccctccccc ttcggatgtg gcttgagctg taggcgcgga
     8041 gtcctgcagc cccgataaaa taaaagattt tatttagtct ccagaaaaag gggggaatga
     8101 aagaccccac ctgtaggttt ggcaagctag ctgcagtaac gccattttgc aaggcatgga
     8161 aaaataccaa accaagaata gagaagttca gatcaagggc gggtacatga aaatagctaa
     8221 cgttgggcca aacaggatat ctgcggtgag cagtttcggc cccggcccgg ggccaagaac
     8281 agatggtcac cgcagtttcg gccccggccc gaggccaaga acagatggtc cccagatatg
     8341 gcccaaccct cagcagtttc ttaagaccca tcagatgttt ccaggctccc ccaaggacct
     8401 gaaatgaccc tgcgccttat ttgaattaac caatcagcct gcttctcgct tctgttcgcg
     8461 cgcttctgct tcccgagctc tataaaagag ctcacaaccc ctcactcggc gcgccagtcc
     8521 tccgacagac tgagtcgccc gggggggatc tggagctctc gagaattctc acgcgtctgc
     8581 aggatatcaa gcttgcggta ccgcgggccc ggccaccatg aacaattccc agggaagagt
     8641 gaccttcgag gatgtcactg tgaacttcac ccagggggag tggcagcggc tgaatcccga
     8701 acagagaaac ttgtacaggg atgtgatgct ggagaattac agcaaccttg tctctgtggg
     8761 acaaggggaa accaccaaac ccgatgtgat cttgaggttg gaacaaggaa aggagccatg
     8821 gttggaggaa gaggaagtgc tgggaagtgg ccgtgcagaa aaaaatgggg acattggagg
     8881 gcagatttgg aagccaaagg atgtgaaaga gagtctctac ccaactttct tgtacaaagt
     8941 ggttggtgga tctggaggtt ctcccaagaa gaagcgcaag gtgggacgcg tctgcaggat
     9001 atcaagcttg cggtaccgcg ggcccgggat cgccaccatg gacaagaagt acagcatcgg
     9061 cctggccatc ggcaccaact ctgtgggctg ggccgtgatc accgacgagt acaaggtgcc
     9121 cagcaagaaa ttcaaggtgc tgggcaacac cgaccggcac agcatcaaga agaacctgat
     9181 cggcgccctg ctgttcgaca gcggagaaac agccgaggcc acccggctga agagaaccgc
     9241 cagaagaaga tacaccagac ggaagaaccg gatctgctat ctgcaagaga tcttcagcaa
     9301 cgagatggcc aaggtggacg acagcttctt ccacagactg gaagagtcct tcctggtgga
     9361 agaggataag aagcacgagc ggcaccccat cttcggcaac atcgtggacg aggtggccta
     9421 ccacgagaag taccccacca tctaccacct gagaaagaaa ctggtggaca gcaccgacaa
     9481 ggccgacctg cggctgatct atctggccct ggcccacatg atcaagttcc ggggccactt
     9541 cctgatcgag ggcgacctga accccgacaa cagcgacgtg gacaagctgt tcatccagct
     9601 ggtgcagacc tacaaccagc tgttcgagga aaaccccatc aacgccagcg gcgtggacgc
     9661 caaggccatc ctgtctgcca gactgagcaa gagcagacgg ctggaaaatc tgatcgccca
     9721 gctgcccggc gagaagaaga atggcctgtt cggcaacctg attgccctga gcctgggcct
     9781 gacccccaac ttcaagagca acttcgacct ggccgaggat gccaaactgc agctgagcaa
     9841 ggacacctac gacgacgacc tggacaacct gctggcccag atcggcgacc agtacgccga
     9901 cctgtttctg gccgccaaga acctgtccga cgccatcctg ctgagcgaca tcctgagagt
     9961 gaacaccgag atcaccaagg cccccctgag cgcctctatg atcaagagat acgacgagca
    10021 ccaccaggac ctgaccctgc tgaaagctct cgtgcggcag cagctgcctg agaagtacaa
    10081 agagattttc ttcgaccaga gcaagaacgg ctacgccggc tacatcgatg gcggagccag
    10141 ccaggaagag ttctacaagt tcatcaagcc catcctggaa aagatggacg gcaccgagga
    10201 actgctcgtg aagctgaaca gagaggacct gctgcggaag cagcggacct tcgacaacgg
    10261 cagcatcccc caccagatcc acctgggaga gctgcacgcc attctgcggc ggcaggaaga
    10321 tttttaccca ttcctgaagg acaaccggga aaagatcgag aagatcctga ccttccgcat
    10381 cccctactac gtgggccctc tggccagggg aaacagcaga ttcgcctgga tgaccagaaa
    10441 gagcgaggaa accatcaccc cctggaactt cgaggaagtg gtggacaagg gcgccagcgc
    10501 ccagagcttc atcgagcgga tgaccaactt cgataagaac ctgcccaacg agaaggtgct
    10561 gcccaagcac agcctgctgt acgagtactt caccgtgtac aacgagctga ccaaagtgaa
    10621 atacgtgacc gagggaatga gaaagcccgc cttcctgagc ggcgagcaga aaaaagccat
    10681 cgtggacctg ctgttcaaga ccaaccggaa agtgaccgtg aagcagctga aagaggacta
    10741 cttcaagaaa atcgagtgct tcgactccgt ggaaatctcc ggcgtggaag atcggttcaa
    10801 cgcctccctg ggcacatacc acgatctgct gaaaattatc aaggacaagg acttcctgga
    10861 caatgaggaa aacgaggaca ttctggaaga tatcgtgctg accctgacac tgtttgagga
    10921 cagagagatg atcgaggaac ggctgaaaac ctatgcccac ctgttcgacg acaaagtgat
    10981 gaagcagctg aagcggcgga gatacaccgg ctggggcagg ctgagccgga agctgatcaa
    11041 cggcatccgg gacaagcagt ccggcaagac aatcctggat ttcctgaagt ccgacggctt
    11101 cgccaacaga aacttcatgc agctgatcca cgacgacagc ctgaccttta aagaggacat
    11161 ccagaaagcc caggtgtccg gccagggcga tagcctgcac gagcacattg ccaatctggc
    11221 cggcagcccc gccattaaga agggcatcct gcagacagtg aaggtggtgg acgagctcgt
    11281 gaaagtgatg ggccggcaca agcccgagaa catcgtgatc gaaatggcca gagagaacca
    11341 gaccacccag aagggacaga agaacagccg cgagagaatg aagcggatcg aagagggcat
    11401 caaagagctg ggcagccaga tcctgaaaga acaccccgtg gaaaacaccc agctgcagaa
    11461 cgagaagctg tacctgtact acctgcagaa tgggcgggat atgtacgtgg accaggaact
    11521 ggacatcaac cggctgtccg actacgatgt ggacgctatc gtgcctcaga gctttctgaa
    11581 ggacgactcc atcgataaca aagtgctgac tcggagcgac aagaaccggg gcaagagcga
    11641 caacgtgccc tccgaagagg tcgtgaagaa gatgaagaac tactggcgcc agctgctgaa
    11701 tgccaagctg attacccaga ggaagttcga caatctgacc aaggccgaga gaggcggcct
    11761 gagcgaactg gataaggccg gcttcatcaa gagacagctg gtggaaaccc ggcagatcac
    11821 aaagcacgtg gcacagatcc tggactcccg gatgaacact aagtacgacg agaacgacaa
    11881 actgatccgg gaagtgaaag tgatcaccct gaagtccaag ctggtgtccg atttccggaa
    11941 ggatttccag ttttacaaag tgcgcgagat caacaactac caccacgccc acgacgccta
    12001 cctgaacgcc gtcgtgggaa ccgccctgat caaaaagtac cctaagctgg aaagcgagtt
    12061 cgtgtacggc gactacaagg tgtacgacgt gcggaagatg atcgccaaga gcgagcagga
    12121 aatcggcaag gctaccgcca agtacttctt ctacagcaac atcatgaact ttttcaagac
    12181 cgagattacc ctggccaacg gcgagatccg gaagcggcct ctgatcgaga caaacggcga
    12241 aacaggcgag atcgtgtggg ataagggccg ggactttgcc accgtgcgga aagtgctgtc
    12301 tatgccccaa gtgaatatcg tgaaaaagac cgaggtgcag acaggcggct tcagcaaaga
    12361 gtctatcctg cccaagagga acagcgacaa gctgatcgcc agaaagaagg actgggaccc
    12421 taagaagtac ggcggcttcg acagccccac cgtggcctat tctgtgctgg tggtggccaa
    12481 agtggaaaag ggcaagtcca agaaactgaa gagtgtgaaa gagctgctgg ggatcaccat
    12541 catggaaaga agcagcttcg agaagaatcc catcgacttt ctggaagcca agggctacaa
    12601 agaagtgaaa aaggacctga tcatcaagct gcctaagtac tccctgttcg agctggaaaa
    12661 cggccggaag agaatgctgg cctctgccgg cgaactgcag aagggaaacg aactggccct
    12721 gccctccaaa tatgtgaact tcctgtacct ggccagccac tatgagaagc tgaagggctc
    12781 ccccgaggat aatgagcaga aacagctgtt tgtggaacag cacaaacact acctggacga
    12841 gatcatcgag cagatcagcg agttctccaa gagagtgatc ctggccgacg ctaatctgga
    12901 caaggtgctg agcgcctaca acaagcacag agacaagcct atcagagagc aggccgagaa
    12961 tatcatccac ctgtttaccc tgaccaatct gggagcccct gccgccttca agtactttga
    13021 caccaccatc gaccggaaga ggtacaccag caccaaagag gtgctggacg ccaccctgat
    13081 ccaccagagc atcaccggcc tgtacgagac acggatcgac ctgtctcagc tgggaggcga
    13141 cgcctatccc tatgacgtgc ccgattatgc cagcctgggc agcggctccc ccaagaaaaa
    13201 acgcaaggtg gaagatccta agaaaaagcg gaaagtgagc ggccgcgcta ctaacttcag
    13261 cctgctgaag caggctggag acgtggagga gaaccctgga cctaagaaac ctgaactgac
    13321 agcaacttct gttgagaagt ttctcattga aaaatttgat tctgtttctg atctcatgca
    13381 gctgtctgaa ggtgaagaaa gcagagcctt ttcttttgat gttggaggaa gaggttatgt
    13441 tctgagggtc aattcttgtg ctgatggttt ttacaaagac agatatgttt acagacactt
    13501 tgcctctgct gctctgccaa ttccagaagt tctggacatt ggagaatttt ctgaatctct
    13561 cacctactgc atcagcagaa gagcacaagg agtcactctc caggatctcc ctgaaactga
    13621 gctgccagct gttctgcaac ctgttgctga agcaatggat gccattgcag cagctgatct
    13681 gagccaaacc tctggatttg gtccttttgg tccccaaggc attggtcagt acaccacttg
    13741 gagggatttc atttgtgcca ttgctgatcc tcatgtctat cactggcaga ctgtgatgga
    13801 tgacacagtt tctgcttctg ttgctcaggc actggatgaa ctcatgctgt gggcagaaga
    13861 ttgtcctgaa gtcagacacc tggtccatgc tgattttgga agcaacaatg ttctgacaga
    13921 caatggcaga atcactgcag tcattgactg gtctgaagcc atgtttggag attctcaata
    13981 tgaggttgcc aacatttttt tttggagacc ttggctggct tgcatggaac aacaaacaag
    14041 atattttgaa agaagacacc cagaactggc tggttccccc agactgagag cctacatgct
    14101 cagaattggc ctggaccaac tgtatcaatc tctggttgat ggaaactttg atgatgctgc
    14161 ttgggcacaa ggaagatgtg atgccattgt gaggtctggt gctggaactg ttggaagaac
    14221 tcaaattgca agaaggtctg ctgctgtttg gactgatgga tgtgttgaag ttctggctga
    14281 ctctggaaac aggagaccct ccacaagacc cagagccaag gaataataag cgatcgccct
    14341 gcaggcatgc aagcttgata tcaagcttat cgataatcaa cctctggatt acaaaatttg
    14401 tgaaagattg actggtattc ttaactatgt tgctcctttt acgctatgtg gatacgctgc
    14461 tttaatgcct ttgtatcatg ctattgcttc ccgtatggct ttcattttct cctccttgta
    14521 taaatcctgg ttgctgtctc tttatgagga gttgtggccc gttgtcaggc aacgtggcgt
    14581 ggtgtgcact gtgtttgctg acgcaacccc cactggttgg ggcattgcca ccacctgtca
    14641 gctcctttcc gggactttcg ctttccccct ccctattgcc acggcggaac tcatcgccgc
    14701 ctgccttgcc cgctgctgga caggggctcg gctgttgggc actgacaatt ccgtggtgtt
    14761 gtcggggaaa tcatcgtcct ttccttggct gctcgcctgt gttgccacct ggattctgcg
    14821 cgggacgtcc ttctgctacg tcccttcggc cctcaatcca gcggaccttc cttcccgcgg
    14881 cctgctgccg gctctgcggc ctcttccgcg tcttcgcctt cgccctcaga cgagtcggat
    14941 ctccctttgg gccgcctccc cgcatcgata ccgtcgacct cgagggaatt aattcgagct
    15001 cggtaccttt aagaccaatg acttacaagg cagctgtaga tcttagccac tttttaaaag
    15061 aaaagggggg actggaaggg ctaattcact cccaacgaag acaagatctg ctttttgctt
    15121 gtactgggtc tctctggtta gaccagatct gagcctggga gctctctggc taactaggga
    15181 acccactgct taagcctcaa taaagcttgc cttgagtgct tcaagtagtg tgtgcccgtc
    15241 tgttgtgtga ctctggtaac tagagatccc tcagaccctt ttag
//
