LOCUS       sequence-372712-       15671 bp    DNA     circular SYN 14-APR-2025
DEFINITION  dCas9 with a C-term NLS-KRAB(Kox1)-MeCP2 fusion (no linker), and GFP
            linked by a P2A site from a SFFV promoter with an upstream 
            ubiquitous chromatin opening element.
ACCESSION   .
VERSION     .
KEYWORDS    .
SOURCE      synthetic DNA construct
  ORGANISM  synthetic DNA construct
REFERENCE   1  (bases 1 to 15671)
  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 15671)
  AUTHORS   .
  TITLE     Direct Submission
  JOURNAL   Exported Apr 14, 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-dCas9-KRAB(Kox1)-MeCP2-P2A-EGFP
FEATURES             Location/Qualifiers
     source          1..15671
                     /mol_type="other DNA"
                     /organism="synthetic DNA construct"
     promoter        complement(48..66)
                     /label=SP6 promoter
                     /note="promoter for bacteriophage SP6 RNA polymerase"
     primer_bind     complement(49..66)
                     /label=SP6
                     /note="SP6 promoter, forward primer"
     primer_bind     complement(356..375)
                     /label=pBRrevBam
                     /note="pBR322 vectors, tet region, downstream of BamHI, 
                     reverse primer"
     primer_bind     complement(589..608)
                     /label=pRS-marker
                     /note="pRS vectors, use to sequence yeast selectable 
                     marker"
     primer_bind     708..730
                     /label=pGEX 3'
                     /note="pGEX vectors, reverse primer"
     primer_bind     complement(768..786)
                     /label=pBRforEco
                     /note="pBR322 vectors, upsteam of EcoRI site, forward 
                     primer"
     promoter        854..958
                     /gene="bla"
                     /label=AmpR promoter
     CDS             join(959..1027,1028..1819)
                     /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(1177..1196)
                     /label=Amp-R
                     /note="Ampicillin resistance gene, reverse primer"
     rep_origin      1990..2578
                     /direction=RIGHT
                     /label=ori
                     /note="high-copy-number ColE1/pMB1/pBR322/pUC origin of 
                     replication"
     primer_bind     2479..2498
                     /label=pBR322ori-F
                     /note="pBR322 origin, forward primer"
     primer_bind     2732..2749
                     /label=L4440
                     /note="L4440 vector, forward primer"
     primer_bind     complement(2819..2839)
                     /label=pBABE 3'
                     /note="SV40 enhancer, reverse primer for pBABE vectors"
     promoter        2824..3153
                     /label=SV40 promoter
                     /note="SV40 enhancer and early promoter"
     rep_origin      3004..3139
                     /label=SV40 ori
                     /note="SV40 origin of replication"
     intron          4287..4352
                     /label=small t intron
                     /note="SV40 (simian virus 40) small t antigen intron"
     CDS             4482..4502
                     /codon_start=1
                     /product="nuclear localization signal of SV40 (simian virus
                     40) large T antigen"
                     /label=SV40 NLS
                     /translation="PKKKRKV"
     polyA_signal    4927..5061
                     /label=SV40 poly(A) signal
                     /note="SV40 polyadenylation signal"
     primer_bind     complement(4964..4983)
                     /label=SV40pA-R
                     /note="SV40 polyA, reverse primer"
     primer_bind     5018..5037
                     /label=EBV-rev
                     /note="SV40 polyA terminator, reverse primer"
     LTR             5229..5862
                     /label=3' LTR
                     /note="3' long terminal repeat (LTR) from HIV-1"
     misc_feature    5909..6034
                     /label=HIV-1 Psi
                     /note="packaging signal of human immunodeficiency virus 
                     type 1"
     misc_feature    6531..6764
                     /label=RRE
                     /note="The Rev response element (RRE) of HIV-1 allows for 
                     Rev-dependent mRNA export from the nucleus to the 
                     cytoplasm."
     CDS             6949..6993
                     /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    7278..7395
                     /label=cPPT/CTS
                     /note="central polypurine tract and central termination 
                     sequence of HIV-1"
     promoter        8137..8544
                     /label=SFFV promoter
                     /note="spleen focus-forming virus long terminal repeat 
                     (LTR) promoter"
     regulatory      8613..8622
                     /label=Kozak sequence
                     /note="vertebrate consensus sequence for strong initiation 
                     of translation (Kozak, 1987)"
                     /regulatory_class="other"
     CDS             join(8619..8645,8646..8648,8649..11132,11133..11138,
                     11139..11204,11205..11207,11208..12722)
                     /codon_start=1
                     /product="catalytically dead mutant of the Cas9 
                     endonuclease from the Streptococcus pyogenes Type II 
                     CRISPR/Cas system"
                     /label=Cas9m4
                     /note="RNA-guided DNA-binding protein that lacks 
                     endonuclease activity due to the mutations D10A, D839A, 
                     H840A, and N863A (Mali et al., 2013)"
                     /translation="MDKKYSIGLAIGTNSVGWAVITDEYKVPSKKFKVLGNTDRHSIKK
                     NLIGALLFDSGETAEATRLKRTARRRYTRRKNRICYLQEIFSNEMAKVDDSFFHRLEES
                     FLVEEDKKHERHPIFGNIVDEVAYHEKYPTIYHLRKKLVDSTDKADLRLIYLALAHMIK
                     FRGHFLIEGDLNPDNSDVDKLFIQLVQTYNQLFEENPINASGVDAKAILSARLSKSRRL
                     ENLIAQLPGEKKNGLFGNLIALSLGLTPNFKSNFDLAEDAKLQLSKDTYDDDLDNLLAQ
                     IGDQYADLFLAAKNLSDAILLSDILRVNTEITKAPLSASMIKRYDEHHQDLTLLKALVR
                     QQLPEKYKEIFFDQSKNGYAGYIDGGASQEEFYKFIKPILEKMDGTEELLVKLNREDLL
                     RKQRTFDNGSIPHQIHLGELHAILRRQEDFYPFLKDNREKIEKILTFRIPYYVGPLARG
                     NSRFAWMTRKSEETITPWNFEEVVDKGASAQSFIERMTNFDKNLPNEKVLPKHSLLYEY
                     FTVYNELTKVKYVTEGMRKPAFLSGEQKKAIVDLLFKTNRKVTVKQLKEDYFKKIECFD
                     SVEISGVEDRFNASLGTYHDLLKIIKDKDFLDNEENEDILEDIVLTLTLFEDREMIEER
                     LKTYAHLFDDKVMKQLKRRRYTGWGRLSRKLINGIRDKQSGKTILDFLKSDGFANRNFM
                     QLIHDDSLTFKEDIQKAQVSGQGDSLHEHIANLAGSPAIKKGILQTVKVVDELVKVMGR
                     HKPENIVIEMARENQTTQKGQKNSRERMKRIEEGIKELGSQILKEHPVENTQLQNEKLY
                     LYYLQNGRDMYVDQELDINRLSDYDVAAIVPQSFLKDDSIDNKVLTRSDKARGKSDNVP
                     SEEVVKKMKNYWRQLLNAKLITQRKFDNLTKAERGGLSELDKAGFIKRQLVETRQITKH
                     VAQILDSRMNTKYDENDKLIREVKVITLKSKLVSDFRKDFQFYKVREINNYHHAHDAYL
                     NAVVGTALIKKYPKLESEFVYGDYKVYDVRKMIAKSEQEIGKATAKYFFYSNIMNFFKT
                     EITLANGEIRKRPLIETNGETGEIVWDKGRDFATVRKVLSMPQVNIVKKTEVQTGGFSK
                     ESILPKRNSDKLIARKKDWDPKKYGGFDSPTVAYSVLVVAKVEKGKSKKLKSVKELLGI
                     TIMERSSFEKNPIDFLEAKGYKEVKKDLIIKLPKYSLFELENGRKRMLASAGELQKGNE
                     LALPSKYVNFLYLASHYEKLKGSPEDNEQKQLFVEQHKHYLDEIIEQISEFSKRVILAD
                     ANLDKVLSAYNKHRDKPIREQAENIIHLFTLTNLGAPAAFKYFDTTIDRKRYTSTKEVL
                     DATLIHQSITGLYETRIDLSQLGGD"
     CDS             12735..12755
                     /codon_start=1
                     /product="nuclear localization signal of SV40 (simian virus
                     40) large T antigen"
                     /label=SV40 NLS
                     /translation="PKKKRKV"
     CDS             12816..13010
                     /codon_start=1
                     /product="Kruppel-associated box (KRAB) transcriptional 
                     repression domain from the human zinc finger protein ZNF10 
                     (Margolin et al., 1994)"
                     /label=KRAB
                     /translation="RTLVTFKDVFVDFTREEWKLLDTAQQIVYRNVMLENYKNLVSLGY
                     QLTKPDVILRLEKGEEPWLV"
     CDS             13044..13064
                     /codon_start=1
                     /product="nuclear localization signal of SV40 (simian virus
                     40) large T antigen"
                     /label=SV40 NLS
                     /translation="PKKKRKV"
     CDS             13074..13931
                     /codon_start=1
                     /product="transcriptional repression domain from rat 
                     methyl-CpG-binding protein 2 (Nan et al., 1997)"
                     /label=MeCP2
                     /translation="VQVKRVLEKSPGKLLVKMPFQASPGGKGEGGGATTSAQVMVIKRP
                     GRKRKAEADPQAIPKKRGRKPGSVVAAAAAEAKKKAVKESSIRSVQETVLPIKKRKTRE
                     TVSIEVKEVVKPLLVSTLGEKSGKGLKTCKSPGRKSKESSPKGRSSSASSPPKKEHHHH
                     HHHAESPKAPMPLLPPPPPPEPQSSEDPISPPEPQDLSSSICKEEKMPRAGSLESDGCP
                     KEPAKTQPMVAAAATTTTTTTTTVAEKYKHRGEGERKDIVSSSMPRPNREEPVDSRTPV
                     TERVS"
     CDS             13941..13997
                     /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(13998..14000,14001..14714)
                     /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(14040..14061)
                     /label=EGFP-N
                     /note="EGFP, reverse primer"
     primer_bind     complement(14301..14320)
                     /label=EXFP-R
                     /note="For distinguishing EGFP variants, reverse primer"
     primer_bind     14648..14669
                     /label=EGFP-C
                     /note="EGFP, forward primer"
     misc_feature    14763..15351
                     /label=WPRE
                     /note="woodchuck hepatitis virus posttranscriptional 
                     regulatory element"
     primer_bind     complement(14816..14836)
                     /label=WPRE-R
                     /note="WPRE, reverse primer"
     CDS             complement(15234..15245)
                     /codon_start=1
                     /product="Factor Xa recognition and cleavage site"
                     /label=Factor Xa site
                     /translation="IEGR"
     primer_bind     complement(15354..15370)
                     /label=KS primer
                     /note="common sequencing primer, one of multiple similar 
                     variants"
     primer_bind     complement(15355..15371)
                     /label=pBluescriptKS
                     /note="For pBluescript vector"
     LTR             join(15461..15671,1..23)
                     /label=3' LTR (Delta-U3)
                     /note="self-inactivating 3' long terminal repeat (LTR) from
                     HIV-1"
ORIGIN
        1 gtcagtgtgg aaaatctcta gcagcatcta gaattaattc cgtgtattct atagtgtcac
       61 ctaaatcgta tgtgtatgat acataaggtt atgtattaat tgtagccgcg ttctaacgac
      121 aatatgtaca agcctaattg tgtagcatct ggcttactga agcagaccct atcatctctc
      181 tcgtaaactg ccgtcagagt cggtttggtt ggacgaacct tctgagtttc tggtaacgcc
      241 gtcccgcacc cggaaatggt cagcgaacca atcagcaggg tcatcgctag ccagatcctc
      301 tacgccggac gcatcgtggc cggcatcacc ggcgccacag gtgcggttgc tggcgcctat
      361 atcgccgaca tcaccgatgg ggaagatcgg gctcgccact tcgggctcat gagcgcttgt
      421 ttcggcgtgg gtatggtggc aggccccgtg gccgggggac tgttgggcgc catctccttg
      481 catgcaccat tccttgcggc ggcggtgctc aacggcctca acctactact gggctgcttc
      541 ctaatgcagg agtcgcataa gggagagcgt cgaatggtgc actctcagta caatctgctc
      601 tgatgccgca tagttaagcc agccccgaca cccgccaaca cccgctgacg cgccctgacg
      661 ggcttgtctg ctcccggcat ccgcttacag acaagctgtg accgtctccg ggagctgcat
      721 gtgtcagagg ttttcaccgt catcaccgaa acgcgcgaga cgaaagggcc tcgtgatacg
      781 cctattttta taggttaatg tcatgataat aatggtttct tagacgtcag gtggcacttt
      841 tcggggaaat gtgcgcggaa cccctatttg tttatttttc taaatacatt caaatatgta
      901 tccgctcatg agacaataac cctgataaat gcttcaataa tattgaaaaa ggaagagtat
      961 gagtattcaa catttccgtg tcgcccttat tccctttttt gcggcatttt gccttcctgt
     1021 ttttgctcac ccagaaacgc tggtgaaagt aaaagatgct gaagatcagt tgggtgcacg
     1081 agtgggttac atcgaactgg atctcaacag cggtaagatc cttgagagtt ttcgccccga
     1141 agaacgtttt ccaatgatga gcacttttaa agttctgcta tgtggcgcgg tattatcccg
     1201 tattgacgcc gggcaagagc aactcggtcg ccgcatacac tattctcaga atgacttggt
     1261 tgagtactca ccagtcacag aaaagcatct tacggatggc atgacagtaa gagaattatg
     1321 cagtgctgcc ataaccatga gtgataacac tgcggccaac ttacttctga caacgatcgg
     1381 aggaccgaag gagctaaccg cttttttgca caacatgggg gatcatgtaa ctcgccttga
     1441 tcgttgggaa ccggagctga atgaagccat accaaacgac gagcgtgaca ccacgatgcc
     1501 tgtagcaatg gcaacaacgt tgcgcaaact attaactggc gaactactta ctctagcttc
     1561 ccggcaacaa ttaatagact ggatggaggc ggataaagtt gcaggaccac ttctgcgctc
     1621 ggcccttccg gctggctggt ttattgctga taaatctgga gccggtgagc gtgggtctcg
     1681 cggtatcatt gcagcactgg ggccagatgg taagccctcc cgtatcgtag ttatctacac
     1741 gacggggagt caggcaacta tggatgaacg aaatagacag atcgctgaga taggtgcctc
     1801 actgattaag cattggtaac tgtcagacca agtttactca tatatacttt agattgattt
     1861 aaaacttcat ttttaattta aaaggatcta ggtgaagatc ctttttgata atctcatgac
     1921 caaaatccct taacgtgagt tttcgttcca ctgagcgtca gaccccgtag aaaagatcaa
     1981 aggatcttct tgagatcctt tttttctgcg cgtaatctgc tgcttgcaaa caaaaaaacc
     2041 accgctacca gcggtggttt gtttgccgga tcaagagcta ccaactcttt ttccgaaggt
     2101 aactggcttc agcagagcgc agataccaaa tactgttctt ctagtgtagc cgtagttagg
     2161 ccaccacttc aagaactctg tagcaccgcc tacatacctc gctctgctaa tcctgttacc
     2221 agtggctgct gccagtggcg ataagtcgtg tcttaccggg ttggactcaa gacgatagtt
     2281 accggataag gcgcagcggt cgggctgaac ggggggttcg tgcacacagc ccagcttgga
     2341 gcgaacgacc tacaccgaac tgagatacct acagcgtgag ctatgagaaa gcgccacgct
     2401 tcccgaaggg agaaaggcgg acaggtatcc ggtaagcggc agggtcggaa caggagagcg
     2461 cacgagggag cttccagggg gaaacgcctg gtatctttat agtcctgtcg ggtttcgcca
     2521 cctctgactt gagcgtcgat ttttgtgatg ctcgtcaggg gggcggagcc tatggaaaaa
     2581 cgccagcaac gcggcctttt tacggttcct ggccttttgc tggccttttg ctcacatgtt
     2641 ctttcctgcg ttatcccctg attctgtgga taaccgtatt accgcctttg agtgagctga
     2701 taccgctcgc cgcagccgaa cgaccgagcg cagcgagtca gtgagcgagg aagcggaaga
     2761 gcgcccaata cgcaaaccgc ctctccccgc gcgttggccg attcattaat gcagctgtgg
     2821 aatgtgtgtc agttagggtg tggaaagtcc ccaggctccc cagcaggcag aagtatgcaa
     2881 agcatgcatc tcaattagtc agcaaccagg tgtggaaagt ccccaggctc cccagcaggc
     2941 agaagtatgc aaagcatgca tctcaattag tcagcaacca tagtcccgcc cctaactccg
     3001 cccatcccgc ccctaactcc gcccagttcc gcccattctc cgccccatgg ctgactaatt
     3061 ttttttattt atgcagaggc cgaggccgcc tcggcctctg agctattcca gaagtagtga
     3121 ggaggctttt ttggaggcct aggcttttgc aaaaagcttg gacacaagac aggcttgcga
     3181 gatatgtttg agaataccac tttatcccgc gtcagggaga ggcagtgcgt aaaaagacgc
     3241 ggactcatgt gaaatactgg tttttagtgc gccagatctc tataatctcg cgcaacctat
     3301 tttcccctcg aacacttttt aagccgtaga taaacaggct gggacacttc acatgagcga
     3361 aaaatacatc gtcacctggg acatgttgca gatccatgca cgtaaactcg caagccgact
     3421 gatgccttct gaacaatgga aaggcattat tgccgtaagc cgtggcggtc tgtaccgggt
     3481 gcgttactgg cgcgtgaact gggtattcgt catgtcgata ccgtttgtat ttccagctac
     3541 gatcacgaca accagcgcga gcttaaagtg ctgaaacgcg cagaaggcga tggcgaaggc
     3601 ttcatcgtta ttgatgacct ggtggatacc ggtggtactg cggttgcgat tcgtgaaatg
     3661 tatccaaaag cgcactttgt caccatcttc gcaaaaccgg ctggtcgtcc gctggttgat
     3721 gactatgttg ttgatatccc gcaagatacc tggattgaac agccgtggga tatgggcgtc
     3781 gtattcgtcc cgccaatctc cggtcgctaa tcttttcaac gcctggcact gccgggcgtt
     3841 gttcttttta acttcaggcg ggttacaata gtttccagta agtattctgg aggctgcatc
     3901 catgacacag gcaaacctga gcgaaaccct gttcaaaccc cgctttaaac atcctgaaac
     3961 ctcgacgcta gtccgccgct ttaatcacgg cgcacaaccg cctgtgcagt cggcccttga
     4021 tggtaaaacc atccctcact ggtatcgcat gattaaccgt ctgatgtgga tctggcgcgg
     4081 cattgaccca cgcgaaatcc tcgacgtcca ggcacgtatt gtgatgagcg atgccgaacg
     4141 taccgacgat gatttatacg atacggtgat tggctaccgt ggcggcaact ggatttatga
     4201 gtgggccccg gatctttgtg aaggaacctt acttctgtgg tgtgacataa ttggacaaac
     4261 tacctacaga gatttaaagc tctaaggtaa atataaaatt tttaagtgta taatgtgtta
     4321 aactactgat tctaattgtt tgtgtatttt agattccaac ctatggaact gatgaatggg
     4381 agcagtggtg gaatgccttt aatgaggaaa acctgttttg ctcagaagaa atgccatcta
     4441 gtgatgatga ggctactgct gactctcaac attctactcc tccaaaaaag aagagaaagg
     4501 tagaagaccc caaggacttt ccttcagaat tgctaagttt tttgagtcat gctgtgttta
     4561 gtaatagaac tcttgcttgc tttgctattt acaccacaaa ggaaaaagct gcactgctat
     4621 acaagaaaat tatggaaaaa tattctgtaa cctttataag taggcataac agttataatc
     4681 ataacatact gttttttctt actccacaca ggcatagagt gtctgctatt aataactatg
     4741 ctcaaaaatt gtgtaccttt agctttttaa tttgtaaagg ggttaataag gaatatttga
     4801 tgtatagtgc cttgactaga gatcataatc agccatacca catttgtaga ggttttactt
     4861 gctttaaaaa acctcccaca cctccccctg aacctgaaac ataaaatgaa tgcaattgtt
     4921 gttgttaact tgtttattgc agcttataat ggttacaaat aaagcaatag catcacaaat
     4981 ttcacaaata aagcattttt ttcactgcat tctagttgtg gtttgtccaa actcatcaat
     5041 gtatcttatc atgtctggat caactggata actcaagcta accaaaatca tcccaaactt
     5101 cccaccccat accctattac cactgccaat tacctgtggt ttcatttact ctaaacctgt
     5161 gattcctctg aattattttc attttaaaga aattgtattt gttaaatatg tactacaaac
     5221 ttagtagttg gaagggctaa ttcactccca aagaagacaa gatatccttg atctgtggat
     5281 ctaccacaca caaggctact tccctgatta gcagaactac acaccagggc caggggtcag
     5341 atatccactg acctttggat ggtgctacaa gctagtacca gttgagccag ataaggtaga
     5401 agaggccaat aaaggagaga acaccagctt gttacaccct gtgagcctgc atgggatgga
     5461 tgacccggag agagaagtgt tagagtggag gtttgacagc cgcctagcat ttcatcacgt
     5521 ggcccgagag ctgcatccgg agtacttcaa gaactgctga tatcgagctt gctacaaggg
     5581 actttccgct ggggactttc cagggaggcg tggcctgggc gggactgggg agtggcgagc
     5641 cctcagatcc tgcatataag cagctgcttt ttgcctgtac tgggtctctc tggttagacc
     5701 agatctgagc ctgggagctc tctggctaac tagggaaccc actgcttaag cctcaataaa
     5761 gcttgccttg agtgcttcaa gtagtgtgtg cccgtctgtt gtgtgactct ggtaactaga
     5821 gatccctcag acccttttag tcagtgtgga aaatctctag cagtggcgcc cgaacaggga
     5881 cttgaaagcg aaagggaaac cagaggagct ctctcgacgc aggactcggc ttgctgaagc
     5941 gcgcacggca agaggcgagg ggcggcgact ggtgagtacg ccaaaaattt tgactagcgg
     6001 aggctagaag gagagagatg ggtgcgagag cgtcagtatt aagcggggga gaattagatc
     6061 gcgatgggaa aaaattcggt taaggccagg gggaaagaaa aaatataaat taaaacatat
     6121 agtatgggca agcagggagc tagaacgatt cgcagttaat cctggcctgt tagaaacatc
     6181 agaaggctgt agacaaatac tgggacagct acaaccatcc cttcagacag gatcagaaga
     6241 acttagatca ttatataata cagtagcaac cctctattgt gtgcatcaaa ggatagagat
     6301 aaaagacacc aaggaagctt tagacaagat agaggaagag caaaacaaaa gtaagaccac
     6361 cgcacagcaa gcggccggcc gctgatcttc agacctggag gaggagatat gagggacaat
     6421 tggagaagtg aattatataa atataaagta gtaaaaattg aaccattagg agtagcaccc
     6481 accaaggcaa agagaagagt ggtgcagaga gaaaaaagag cagtgggaat aggagctttg
     6541 ttccttgggt tcttgggagc agcaggaagc actatgggcg cagcgtcaat gacgctgacg
     6601 gtacaggcca gacaattatt gtctggtata gtgcagcagc agaacaattt gctgagggct
     6661 attgaggcgc aacagcatct gttgcaactc acagtctggg gcatcaagca gctccaggca
     6721 agaatcctgg ctgtggaaag atacctaaag gatcaacagc tcctggggat ttggggttgc
     6781 tctggaaaac tcatttgcac cactgctgtg ccttggaatg ctagttggag taataaatct
     6841 ctggaacaga tttggaatca cacgacctgg atggagtggg acagagaaat taacaattac
     6901 acaagcttaa tacactcctt aattgaagaa tcgcaaaacc agcaagaaaa gaatgaacaa
     6961 gaattattgg aattagataa atgggcaagt ttgtggaatt ggtttaacat aacaaattgg
     7021 ctgtggtata taaaattatt cataatgata gtaggaggct tggtaggttt aagaatagtt
     7081 tttgctgtac tttctatagt gaatagagtt aggcagggat attcaccatt atcgtttcag
     7141 acccacctcc caaccccgag gggacccgac aggcccgaag gaatagaaga agaaggtgga
     7201 gagagagaca gagacagatc cattcgatta gtgaacggat ctcgacggta tcgccaaatg
     7261 gcagtattca tccacaattt taaaagaaaa ggggggattg gggggtacag tgcaggggaa
     7321 agaatagtag acataatagc aacagacata caaactaaag aattacaaaa acaaattaca
     7381 aaaattcaaa attttcgggt ttattacagg gacagcagag atccagtttg gatcgataag
     7441 cttgatatcg aattcgggag gtggtccctg cagttacgcc aatgataacc cccgccagaa
     7501 aaatcttagt agccttccct ttttgttttc cgtgccccaa ctcggcggat tgactcggcc
     7561 ccttccggaa acacccgaat caacttctag tcaaattatt gttcacgccg caatgaccca
     7621 cccctggccc gcgtctgtgg aactgacccc tggtgtacag gagagttcgc tgctgaaagt
     7681 ggtcccaaag gggtactagt ttttaagctc ccaactcccc ctcccccagc gtctggagga
     7741 ttccacaccc tcgcaccgca ggggcgagga agtgggcgga gtccggtttt ggcgccagcc
     7801 gctgaggctg ccaagcagaa aagccaccgc tgaggagact ccggtcactg tcctcgcccc
     7861 gcctccccct tccctcccct tggggaccac cgggcgccac gccgcgaacg gtaagtgccg
     7921 cggtcgtcgg cgcctccgcc ctccccctag ggccccaatt cccagcgggc gcggcgcgcg
     7981 gccccgcgcg cagctcccgg ctccctcccc cttcggatgt ggcttgagct gtaggcgcgg
     8041 agtcctgcag ccccgataaa ataaaagatt ttatttagtc tccagaaaaa ggggggaatg
     8101 aaagacccca cctgtaggtt tggcaagcta gctgcagtaa cgccattttg caaggcatgg
     8161 aaaaatacca aaccaagaat agagaagttc agatcaaggg cgggtacatg aaaatagcta
     8221 acgttgggcc aaacaggata tctgcggtga gcagtttcgg ccccggcccg gggccaagaa
     8281 cagatggtca ccgcagtttc ggccccggcc cgaggccaag aacagatggt ccccagatat
     8341 ggcccaaccc tcagcagttt cttaagaccc atcagatgtt tccaggctcc cccaaggacc
     8401 tgaaatgacc ctgcgcctta tttgaattaa ccaatcagcc tgcttctcgc ttctgttcgc
     8461 gcgcttctgc ttcccgagct ctataaaaga gctcacaacc cctcactcgg cgcgccagtc
     8521 ctccgacaga ctgagtcgcc cgggggggat ctggagctct cgagaattct cacgcgtctg
     8581 caggatatca agcttgcggt accgcgggcc cggccaccat ggacaagaag tactccattg
     8641 ggctcgctat cggcacaaac agcgtcggct gggccgtcat tacggacgag tacaaggtgc
     8701 cgagcaaaaa attcaaagtt ctgggcaata ccgatcgcca cagcataaag aagaacctca
     8761 ttggcgccct cctgttcgac tccggggaga cggccgaagc cacgcggctc aaaagaacag
     8821 cacggcgcag atatacccgc agaaagaatc ggatctgcta cctgcaggag atctttagta
     8881 atgagatggc taaggtggat gactctttct tccataggct ggaggagtcc tttttggtgg
     8941 aggaggataa aaagcacgag cgccacccaa tctttggcaa tatcgtggac gaggtggcgt
     9001 accatgaaaa gtacccaacc atatatcatc tgaggaagaa gcttgtagac agtactgata
     9061 aggctgactt gcggttgatc tatctcgcgc tggcgcatat gatcaaattt cggggacact
     9121 tcctcatcga gggggacctg aacccagaca acagcgatgt cgacaaactc tttatccaac
     9181 tggttcagac ttacaatcag cttttcgaag agaacccgat caacgcatcc ggagttgacg
     9241 ccaaagcaat cctgagcgct aggctgtcca aatcccggcg gctcgaaaac ctcatcgcac
     9301 agctccctgg ggagaagaag aacggcctgt ttggtaatct tatcgccctg tcactcgggc
     9361 tgacccccaa ctttaaatct aacttcgacc tggccgaaga tgccaagctt caactgagca
     9421 aagacaccta cgatgatgat ctcgacaatc tgctggccca gatcggcgac cagtacgcag
     9481 accttttttt ggcggcaaag aacctgtcag acgccattct gctgagtgat attctgcgag
     9541 tgaacacgga gatcaccaaa gctccgctga gcgctagtat gatcaagcgc tatgatgagc
     9601 accaccaaga cttgactttg ctgaaggccc ttgtcagaca gcaactgcct gagaagtaca
     9661 aggaaatttt cttcgatcag tctaaaaatg gctacgccgg atacattgac ggcggagcaa
     9721 gccaggagga attttacaaa tttattaagc ccatcttgga aaaaatggac ggcaccgagg
     9781 agctgctggt aaagcttaac agagaagatc tgttgcgcaa acagcgcact ttcgacaatg
     9841 gaagcatccc ccaccagatt cacctgggcg aactgcacgc tatcctcagg cggcaagagg
     9901 atttctaccc ctttttgaaa gataacaggg aaaagattga gaaaatcctc acatttcgga
     9961 taccctacta tgtaggcccc ctcgcccggg gaaattccag attcgcgtgg atgactcgca
    10021 aatcagaaga gaccatcact ccctggaact tcgaggaagt cgtggataag ggggcctctg
    10081 cccagtcctt catcgaaagg atgactaact ttgataaaaa tctgcctaac gaaaaggtgc
    10141 ttcctaaaca ctctctgctg tacgagtact tcacagttta taacgagctc accaaggtca
    10201 aatacgtcac agaagggatg agaaagccag cattcctgtc tggagagcag aagaaagcta
    10261 tcgtggacct cctcttcaag acgaaccgga aagttaccgt gaaacagctc aaagaagact
    10321 atttcaaaaa gattgaatgt ttcgactctg ttgaaatcag cggagtggag gatcgcttca
    10381 acgcatccct gggaacgtat cacgatctcc tgaaaatcat taaagacaag gacttcctgg
    10441 acaatgagga gaacgaggac attcttgagg acattgtcct cacccttacg ttgtttgaag
    10501 atagggagat gattgaagaa cgcttgaaaa cttacgctca tctcttcgac gacaaagtca
    10561 tgaaacagct caagaggcgc cgatatacag gatgggggcg gctgtcaaga aaactgatca
    10621 atgggatccg agacaagcag agtggaaaga caatcctgga ttttcttaag tccgatggat
    10681 ttgccaaccg gaacttcatg cagttgatcc atgatgactc tctcaccttt aaggaggaca
    10741 tccagaaagc acaagtttct ggccaggggg acagtcttca cgagcacatc gctaatcttg
    10801 caggtagccc agctatcaaa aagggaatac tgcagaccgt taaggtcgtg gatgaactcg
    10861 tcaaagtaat gggaaggcat aagcccgaga atatcgttat cgagatggcc cgagagaacc
    10921 aaactaccca gaagggacag aagaacagta gggaaaggat gaagaggatt gaagagggta
    10981 taaaagaact ggggtcccaa atccttaagg aacacccagt tgaaaacacc cagcttcaga
    11041 atgagaagct ctacctgtac tacctgcaga acggcaggga catgtacgtg gatcaggaac
    11101 tggacatcaa tcggctctcc gactacgacg tggctgctat cgtgccccag tcttttctca
    11161 aagatgattc tattgataat aaagtgttga caagatccga taaagctaga gggaagagtg
    11221 ataacgtccc ctcagaagaa gttgtcaaga aaatgaaaaa ttattggcgg cagctgctga
    11281 acgccaaact gatcacacaa cggaagttcg ataatctgac taaggctgaa cgaggtggcc
    11341 tgtctgagtt ggataaagcc ggcttcatca aaaggcagct tgttgagaca cgccagatca
    11401 ccaagcacgt ggcccaaatt ctcgattcac gcatgaacac caagtacgat gaaaatgaca
    11461 aactgattcg agaggtgaaa gttattactc tgaagtctaa gctggtctca gatttcagaa
    11521 aggactttca gttttataag gtgagagaga tcaacaatta ccaccatgcg catgatgcct
    11581 acctgaatgc agtggtaggc actgcactta tcaaaaaata tcccaagctt gaatctgaat
    11641 ttgtttacgg agactataaa gtgtacgatg ttaggaaaat gatcgcaaag tctgagcagg
    11701 aaataggcaa ggccaccgct aagtacttct tttacagcaa tattatgaat tttttcaaga
    11761 ccgagattac actggccaat ggagagattc ggaagcgacc acttatcgaa acaaacggag
    11821 aaacaggaga aatcgtgtgg gacaagggta gggatttcgc gacagtccgg aaggtcctgt
    11881 ccatgccgca ggtgaacatc gttaaaaaga ccgaagtaca gaccggaggc ttctccaagg
    11941 aaagtatcct cccgaaaagg aacagcgaca agctgatcgc acgcaaaaaa gattgggacc
    12001 ccaagaaata cggcggattc gattctccta cagtcgctta cagtgtactg gttgtggcca
    12061 aagtggagaa agggaagtct aaaaaactca aaagcgtcaa ggaactgctg ggcatcacaa
    12121 tcatggagcg atcaagcttc gaaaaaaacc ccatcgactt tctcgaggcg aaaggatata
    12181 aagaggtcaa aaaagacctc atcattaagc ttcccaagta ctctctcttt gagcttgaaa
    12241 acggccggaa acgaatgctc gctagtgcgg gcgagctgca gaaaggtaac gagctggcac
    12301 tgccctctaa atacgttaat ttcttgtatc tggccagcca ctatgaaaag ctcaaagggt
    12361 ctcccgaaga taatgagcag aagcagctgt tcgtggaaca acacaaacac taccttgatg
    12421 agatcatcga gcaaataagc gaattctcca aaagagtgat cctcgccgac gctaacctcg
    12481 ataaggtgct ttctgcttac aataagcaca gggataagcc catcagggag caggcagaaa
    12541 acattatcca cttgtttact ctgaccaact tgggcgcgcc tgcagccttc aagtacttcg
    12601 acaccaccat agacagaaag cggtacacct ctacaaagga ggtcctggac gccacactga
    12661 ttcatcagtc aattacgggg ctctatgaaa caagaatcga cctctctcag ctcggtggag
    12721 acagcagggc tgaccccaag aagaagagga aggtgagtgg tggaggaagt ggcgggtcag
    12781 ggtcgatgga cgcgaaatca cttacggcat ggtcgagaac actggttacg ttcaaggacg
    12841 tgtttgtgga ctttacacgt gaggagtgga aattgctgga tactgcgcaa caaattgtgt
    12901 atcgaaatgt catgcttgag aattacaaga acctcgtcag tctcggatac cagttgacga
    12961 aaccggatgt gatccttagg ctcgaaaagg gggaagaacc ttggctggta tcgggaggtg
    13021 gttcgggtgg ctctggatca agcccaaaga agaaacggaa ggtggaagcc tcagtgcagg
    13081 tgaaaagggt gctggaaaaa tcccccggca aactcctcgt gaagatgccc ttccaggctt
    13141 cccctggcgg aaaaggtgaa gggggtggcg caaccacatc tgcccaggtc atggtcatca
    13201 agcgacctgg aaggaaaaga aaggccgagg ctgaccctca ggccattcca aagaaacggg
    13261 gacgcaagcc agggtccgtg gtcgcagctg cagcagctga ggctaagaaa aaggcagtga
    13321 aggaaagctc catccgcagt gtgcaggaga ctgtcctgcc catcaagaag aggaagacta
    13381 gggagaccgt gtccatcgag gtcaaagaag tggtcaagcc cctgctcgtg tccaccctgg
    13441 gcgaaaaatc tggaaagggg ctcaaaacat gcaagtcacc tggacggaaa agcaaggagt
    13501 ctagtccaaa ggggcgctca agctccgctt ctagtccccc taaaaaggaa caccatcacc
    13561 atcaccatca cgccgagtct cctaaggctc ctatgccact gctcccacca cctccaccac
    13621 ctgagccaca gtcaagcgaa gaccccatca gcccacccga gcctcaggat ctgtcctcta
    13681 gtatttgcaa agaggaaaag atgcccagag caggcagcct ggagagtgat ggctgtccaa
    13741 aagaacccgc caagacccag cctatggtgg cagccgctgc aactaccacc acaaccacaa
    13801 ctaccacagt ggccgaaaaa tacaagcatc gcggcgaggg cgaacgaaag gacattgtgt
    13861 caagctccat gcccagacct aaccgggagg aaccagtcga tagtaggaca cccgtgactg
    13921 agagagtctc aagcggccgc gctactaact tcagcctgct gaagcaggct ggagacgtgg
    13981 aggagaaccc tggacctgtg agcaagggcg aggagctgtt caccggggtg gtgcccatcc
    14041 tggtcgagct ggacggcgac gtaaacggcc acaagttcag cgtgtctggc gagggcgagg
    14101 gcgatgccac ctacggcaag ctgaccctga agttcatctg caccaccggc aagctgcccg
    14161 tgccctggcc caccctcgtg accaccctga cctacggcgt gcagtgcttc agccgctacc
    14221 ccgaccacat gaagcagcac gacttcttca agtccgccat gcccgaaggc tacgtccagg
    14281 agcgcaccat cttcttcaag gacgacggca actacaagac ccgcgccgag gtgaagttcg
    14341 agggcgacac cctggtgaac cgcatcgagc tgaagggcat cgacttcaag gaggacggca
    14401 acatcctggg gcacaagctg gagtacaact acaacagcca caacgtctat atcatggccg
    14461 acaagcagaa gaacggcatc aaggcgaact tcaagatccg ccacaacatc gaggacggca
    14521 gcgtgcagct cgccgaccac taccagcaga acacccccat cggcgacggc cccgtgctgc
    14581 tgcccgacaa ccactacctg agcacccagt ccgccctgag caaagacccc aacgagaagc
    14641 gcgatcacat ggtcctgctg gagttcgtga ccgccgccgg gatcactctc ggcatggacg
    14701 agctgtacaa gtaataagcg atcgccctgc aggcatgcaa gcttgatatc aagcttatcg
    14761 ataatcaacc tctggattac aaaatttgtg aaagattgac tggtattctt aactatgttg
    14821 ctccttttac gctatgtgga tacgctgctt taatgccttt gtatcatgct attgcttccc
    14881 gtatggcttt cattttctcc tccttgtata aatcctggtt gctgtctctt tatgaggagt
    14941 tgtggcccgt tgtcaggcaa cgtggcgtgg tgtgcactgt gtttgctgac gcaaccccca
    15001 ctggttgggg cattgccacc acctgtcagc tcctttccgg gactttcgct ttccccctcc
    15061 ctattgccac ggcggaactc atcgccgcct gccttgcccg ctgctggaca ggggctcggc
    15121 tgttgggcac tgacaattcc gtggtgttgt cggggaaatc atcgtccttt ccttggctgc
    15181 tcgcctgtgt tgccacctgg attctgcgcg ggacgtcctt ctgctacgtc ccttcggccc
    15241 tcaatccagc ggaccttcct tcccgcggcc tgctgccggc tctgcggcct cttccgcgtc
    15301 ttcgccttcg ccctcagacg agtcggatct ccctttgggc cgcctccccg catcgatacc
    15361 gtcgacctcg agggaattaa ttcgagctcg gtacctttaa gaccaatgac ttacaaggca
    15421 gctgtagatc ttagccactt tttaaaagaa aaggggggac tggaagggct aattcactcc
    15481 caacgaagac aagatctgct ttttgcttgt actgggtctc tctggttaga ccagatctga
    15541 gcctgggagc tctctggcta actagggaac ccactgctta agcctcaata aagcttgcct
    15601 tgagtgcttc aagtagtgtg tgcccgtctg ttgtgtgact ctggtaacta gagatccctc
    15661 agaccctttt a
//
