LOCUS       sequence-354320-       14176 bp    DNA     circular SYN 15-DEC-2025
DEFINITION  Lentiviral expression vector for dCas9-KRAB in mammalian cells.
ACCESSION   .
VERSION     .
KEYWORDS    .
SOURCE      synthetic DNA construct
  ORGANISM  synthetic DNA construct
REFERENCE   1  (bases 1 to 14176)
  AUTHORS   Schmidt R, Steinhart Z, Layeghi M, Freimer JW, Bueno R, Nguyen VQ, 
            Blaeschke F, Ye CJ, Marson A
  TITLE     CRISPR activation and interference screens decode stimulation 
            responses in primary human T cells.
  JOURNAL   Science. 2022 Feb 4;375(6580):eabj4008. doi: 
            10.1126/science.abj4008. Epub 2022 Feb 4.
  PUBMED    35113687
REFERENCE   2  (bases 1 to 14176)
  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: "Science."; 
            date: "2022-02-4"; volume: "375(6580):eabj4008. doi"; pages: " 
            10.1126/science.abj4008. Epub 2022 Feb 4"
COMMENT     Sequence Label: pZR071_SFFV-dCas9-mCherry-KRAB
FEATURES             Location/Qualifiers
     source          1..14176
                     /mol_type="other DNA"
                     /organism="synthetic DNA construct"
     misc_feature    668..793
                     /label=HIV-1 Psi
                     /note="packaging signal of human immunodeficiency virus 
                     type 1"
     misc_feature    1290..1523
                     /label=RRE
                     /note="The Rev response element (RRE) of HIV-1 allows for 
                     Rev-dependent mRNA export from the nucleus to the 
                     cytoplasm."
     CDS             1708..1752
                     /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    2037..2154
                     /label=cPPT/CTS
                     /note="central polypurine tract and central termination 
                     sequence of HIV-1"
     promoter        2307..2714
                     /label=SFFV promoter
                     /note="spleen focus-forming virus long terminal repeat 
                     (LTR) promoter"
     regulatory      2783..2792
                     /label=Kozak sequence
                     /note="vertebrate consensus sequence for strong initiation 
                     of translation (Kozak, 1987)"
                     /regulatory_class="other"
     CDS             join(2789..2815,2816..2818,2819..5305,5306..5308,
                     5309..6892)
                     /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             2789..6892
                     /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             6896..6922
                     /codon_start=1
                     /product="HA (human influenza hemagglutinin) epitope tag"
                     /label=HA
                     /translation="YPYDVPDYA"
     CDS             6902..6928
                     /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             6941..6961
                     /codon_start=1
                     /product="nuclear localization signal of SV40 (simian virus
                     40) large T antigen"
                     /label=SV40 NLS
                     /translation="PKKKRKV"
     CDS             6968..6988
                     /codon_start=1
                     /product="nuclear localization signal of SV40 (simian virus
                     40) large T antigen"
                     /label=SV40 NLS
                     /translation="PKKKRKV"
     CDS             7028..7732
                     /codon_start=1
                     /product="monomeric derivative of DsRed fluorescent protein
                     (Shaner et al., 2004)"
                     /label=mCherry
                     /note="mammalian codon-optimized"
                     /translation="VSKGEEDNMAIIKEFMRFKVHMEGSVNGHEFEIEGEGEGRPYEGT
                     QTAKLKVTKGGPLPFAWDILSPQFMYGSKAYVKHPADIPDYLKLSFPEGFKWERVMNFE
                     DGGVVTVTQDSSLQDGEFIYKVKLRGTNFPSDGPVMQKKTMGWEASSERMYPEDGALKG
                     EIKQRLKLKDGGHYDAEVKTTYKAKKPVQLPGAYNVNIKLDITSHNEDYTIVEQYERAE
                     GRHSTGGMDELYK"
     primer_bind     complement(7170..7188)
                     /label=mCherry-R
                     /note="mCherry, reverse primer"
     primer_bind     7438..7457
                     /label=mCherry-F
                     /note="mCherry, forward primer"
     CDS             7778..7963
                     /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
                     QLTKPDVILRLEKGEEP"
     misc_feature    8027..8615
                     /label=WPRE
                     /note="woodchuck hepatitis virus posttranscriptional 
                     regulatory element"
     primer_bind     complement(8080..8100)
                     /label=WPRE-R
                     /note="WPRE, reverse primer"
     CDS             complement(8498..8509)
                     /codon_start=1
                     /product="Factor Xa recognition and cleavage site"
                     /label=Factor Xa site
                     /translation="IEGR"
     primer_bind     complement(8618..8634)
                     /label=KS primer
                     /note="common sequencing primer, one of multiple similar 
                     variants"
     primer_bind     complement(8619..8635)
                     /label=pBluescriptKS
                     /note="For pBluescript vector"
     LTR             8725..8958
                     /label=3' LTR (Delta-U3)
                     /note="self-inactivating 3' long terminal repeat (LTR) from
                     HIV-1"
     promoter        complement(8983..9001)
                     /label=SP6 promoter
                     /note="promoter for bacteriophage SP6 RNA polymerase"
     primer_bind     complement(8984..9001)
                     /label=SP6
                     /note="SP6 promoter, forward primer"
     primer_bind     complement(9291..9310)
                     /label=pBRrevBam
                     /note="pBR322 vectors, tet region, downstream of BamHI, 
                     reverse primer"
     primer_bind     complement(9524..9543)
                     /label=pRS-marker
                     /note="pRS vectors, use to sequence yeast selectable 
                     marker"
     primer_bind     9643..9665
                     /label=pGEX 3'
                     /note="pGEX vectors, reverse primer"
     primer_bind     complement(9703..9721)
                     /label=pBRforEco
                     /note="pBR322 vectors, upsteam of EcoRI site, forward 
                     primer"
     promoter        9789..9893
                     /gene="bla"
                     /label=AmpR promoter
     CDS             join(9894..9962,9963..10754)
                     /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(10112..10131)
                     /label=Amp-R
                     /note="Ampicillin resistance gene, reverse primer"
     rep_origin      10925..11513
                     /direction=RIGHT
                     /label=ori
                     /note="high-copy-number ColE1/pMB1/pBR322/pUC origin of 
                     replication"
     primer_bind     11414..11433
                     /label=pBR322ori-F
                     /note="pBR322 origin, forward primer"
     primer_bind     11667..11684
                     /label=L4440
                     /note="L4440 vector, forward primer"
     primer_bind     complement(11754..11774)
                     /label=pBABE 3'
                     /note="SV40 enhancer, reverse primer for pBABE vectors"
     promoter        11759..12088
                     /label=SV40 promoter
                     /note="SV40 enhancer and early promoter"
     rep_origin      11939..12074
                     /label=SV40 ori
                     /note="SV40 origin of replication"
     intron          13222..13287
                     /label=small t intron
                     /note="SV40 (simian virus 40) small t antigen intron"
     CDS             13417..13437
                     /codon_start=1
                     /product="nuclear localization signal of SV40 (simian virus
                     40) large T antigen"
                     /label=SV40 NLS
                     /translation="PKKKRKV"
     polyA_signal    13862..13996
                     /label=SV40 poly(A) signal
                     /note="SV40 polyadenylation signal"
     primer_bind     complement(13899..13918)
                     /label=SV40pA-R
                     /note="SV40 polyA, reverse primer"
     primer_bind     13953..13972
                     /label=EBV-rev
                     /note="SV40 polyA terminator, reverse primer"
     LTR             join(14164..14176,1..621)
                     /label=3' LTR
                     /note="3' long terminal repeat (LTR) from HIV-1"
ORIGIN
        1 tcactcccaa agaagacaag atatccttga tctgtggatc taccacacac aaggctactt
       61 ccctgattag cagaactaca caccagggcc aggggtcaga tatccactga cctttggatg
      121 gtgctacaag ctagtaccag ttgagccaga taaggtagaa gaggccaata aaggagagaa
      181 caccagcttg ttacaccctg tgagcctgca tgggatggat gacccggaga gagaagtgtt
      241 agagtggagg tttgacagcc gcctagcatt tcatcacgtg gcccgagagc tgcatccgga
      301 gtacttcaag aactgctgat atcgagcttg ctacaaggga ctttccgctg gggactttcc
      361 agggaggcgt ggcctgggcg ggactgggga gtggcgagcc ctcagatcct gcatataagc
      421 agctgctttt tgcctgtact gggtctctct ggttagacca gatctgagcc tgggagctct
      481 ctggctaact agggaaccca ctgcttaagc ctcaataaag cttgccttga gtgcttcaag
      541 tagtgtgtgc ccgtctgttg tgtgactctg gtaactagag atccctcaga cccttttagt
      601 cagtgtggaa aatctctagc agtggcgccc gaacagggac ttgaaagcga aagggaaacc
      661 agaggagctc tctcgacgca ggactcggct tgctgaagcg cgcacggcaa gaggcgaggg
      721 gcggcgactg gtgagtacgc caaaaatttt gactagcgga ggctagaagg agagagatgg
      781 gtgcgagagc gtcagtatta agcgggggag aattagatcg cgatgggaaa aaattcggtt
      841 aaggccaggg ggaaagaaaa aatataaatt aaaacatata gtatgggcaa gcagggagct
      901 agaacgattc gcagttaatc ctggcctgtt agaaacatca gaaggctgta gacaaatact
      961 gggacagcta caaccatccc ttcagacagg atcagaagaa cttagatcat tatataatac
     1021 agtagcaacc ctctattgtg tgcatcaaag gatagagata aaagacacca aggaagcttt
     1081 agacaagata gaggaagagc aaaacaaaag taagaccacc gcacagcaag cggccggccg
     1141 ctgatcttca gacctggagg aggagatatg agggacaatt ggagaagtga attatataaa
     1201 tataaagtag taaaaattga accattagga gtagcaccca ccaaggcaaa gagaagagtg
     1261 gtgcagagag aaaaaagagc agtgggaata ggagctttgt tccttgggtt cttgggagca
     1321 gcaggaagca ctatgggcgc agcgtcaatg acgctgacgg tacaggccag acaattattg
     1381 tctggtatag tgcagcagca gaacaatttg ctgagggcta ttgaggcgca acagcatctg
     1441 ttgcaactca cagtctgggg catcaagcag ctccaggcaa gaatcctggc tgtggaaaga
     1501 tacctaaagg atcaacagct cctggggatt tggggttgct ctggaaaact catttgcacc
     1561 actgctgtgc cttggaatgc tagttggagt aataaatctc tggaacagat ttggaatcac
     1621 acgacctgga tggagtggga cagagaaatt aacaattaca caagcttaat acactcctta
     1681 attgaagaat cgcaaaacca gcaagaaaag aatgaacaag aattattgga attagataaa
     1741 tgggcaagtt tgtggaattg gtttaacata acaaattggc tgtggtatat aaaattattc
     1801 ataatgatag taggaggctt ggtaggttta agaatagttt ttgctgtact ttctatagtg
     1861 aatagagtta ggcagggata ttcaccatta tcgtttcaga cccacctccc aaccccgagg
     1921 ggacccgaca ggcccgaagg aatagaagaa gaaggtggag agagagacag agacagatcc
     1981 attcgattag tgaacggatc tcgacggtat cgccaaatgg cagtattcat ccacaatttt
     2041 aaaagaaaag gggggattgg ggggtacagt gcaggggaaa gaatagtaga cataatagca
     2101 acagacatac aaactaaaga attacaaaaa caaattacaa aaattcaaaa ttttcgggtt
     2161 tattacaggg acagcagaga tccagtttgg atcgataagc ttgatatcga attcctgcag
     2221 ccccgataaa ataaaagatt ttatttagtc tccagaaaaa ggggggaatg aaagacccca
     2281 cctgtaggtt tggcaagcta gctgcagtaa cgccattttg caaggcatgg aaaaatacca
     2341 aaccaagaat agagaagttc agatcaaggg cgggtacatg aaaatagcta acgttgggcc
     2401 aaacaggata tctgcggtga gcagtttcgg ccccggcccg gggccaagaa cagatggtca
     2461 ccgcagtttc ggccccggcc cgaggccaag aacagatggt ccccagatat ggcccaaccc
     2521 tcagcagttt cttaagaccc atcagatgtt tccaggctcc cccaaggacc tgaaatgacc
     2581 ctgcgcctta tttgaattaa ccaatcagcc tgcttctcgc ttctgttcgc gcgcttctgc
     2641 ttcccgagct ctataaaaga gctcacaacc cctcactcgg cgcgccagtc ctccgacaga
     2701 ctgagtcgcc cgggggggat ctggagctct cgagaattct cacgcgtctg caggatatca
     2761 agcttgcggt accgcgggcc cggccaccat ggacaagaag tacagcatcg gcctggccat
     2821 cggcaccaac tctgtgggct gggccgtgat caccgacgag tacaaggtgc ccagcaagaa
     2881 attcaaggtg ctgggcaaca ccgaccggca cagcatcaag aagaacctga tcggcgccct
     2941 gctgttcgac agcggagaaa cagccgaggc cacccggctg aagagaaccg ccagaagaag
     3001 atacaccaga cggaagaacc ggatctgcta tctgcaagag atcttcagca acgagatggc
     3061 caaggtggac gacagcttct tccacagact ggaagagtcc ttcctggtgg aagaggataa
     3121 gaagcacgag cggcacccca tcttcggcaa catcgtggac gaggtggcct accacgagaa
     3181 gtaccccacc atctaccacc tgagaaagaa actggtggac agcaccgaca aggccgacct
     3241 gcggctgatc tatctggccc tggcccacat gatcaagttc cggggccact tcctgatcga
     3301 gggcgacctg aaccccgaca acagcgacgt ggacaagctg ttcatccagc tggtgcagac
     3361 ctacaaccag ctgttcgagg aaaaccccat caacgccagc ggcgtggacg ccaaggccat
     3421 cctgtctgcc agactgagca agagcagacg gctggaaaat ctgatcgccc agctgcccgg
     3481 cgagaagaag aatggcctgt tcggcaacct gattgccctg agcctgggcc tgacccccaa
     3541 cttcaagagc aacttcgacc tggccgagga tgccaaactg cagctgagca aggacaccta
     3601 cgacgacgac ctggacaacc tgctggccca gatcggcgac cagtacgccg acctgtttct
     3661 ggccgccaag aacctgtccg acgccatcct gctgagcgac atcctgagag tgaacaccga
     3721 gatcaccaag gcccccctga gcgcctctat gatcaagaga tacgacgagc accaccagga
     3781 cctgaccctg ctgaaagctc tcgtgcggca gcagctgcct gagaagtaca aagagatttt
     3841 cttcgaccag agcaagaacg gctacgccgg ctacatcgat ggcggagcca gccaggaaga
     3901 gttctacaag ttcatcaagc ccatcctgga aaagatggac ggcaccgagg aactgctcgt
     3961 gaagctgaac agagaggacc tgctgcggaa gcagcggacc ttcgacaacg gcagcatccc
     4021 ccaccagatc cacctgggag agctgcacgc cattctgcgg cggcaggaag atttttaccc
     4081 attcctgaag gacaaccggg aaaagatcga gaagatcctg accttccgca tcccctacta
     4141 cgtgggccct ctggccaggg gaaacagcag attcgcctgg atgaccagaa agagcgagga
     4201 aaccatcacc ccctggaact tcgaggaagt ggtggacaag ggcgccagcg cccagagctt
     4261 catcgagcgg atgaccaact tcgataagaa cctgcccaac gagaaggtgc tgcccaagca
     4321 cagcctgctg tacgagtact tcaccgtgta caacgagctg accaaagtga aatacgtgac
     4381 cgagggaatg agaaagcccg ccttcctgag cggcgagcag aaaaaagcca tcgtggacct
     4441 gctgttcaag accaaccgga aagtgaccgt gaagcagctg aaagaggact acttcaagaa
     4501 aatcgagtgc ttcgactccg tggaaatctc cggcgtggaa gatcggttca acgcctccct
     4561 gggcacatac cacgatctgc tgaaaattat caaggacaag gacttcctgg acaatgagga
     4621 aaacgaggac attctggaag atatcgtgct gaccctgaca ctgtttgagg acagagagat
     4681 gatcgaggaa cggctgaaaa cctatgccca cctgttcgac gacaaagtga tgaagcagct
     4741 gaagcggcgg agatacaccg gctggggcag gctgagccgg aagctgatca acggcatccg
     4801 ggacaagcag tccggcaaga caatcctgga tttcctgaag tccgacggct tcgccaacag
     4861 aaacttcatg cagctgatcc acgacgacag cctgaccttt aaagaggaca tccagaaagc
     4921 ccaggtgtcc ggccagggcg atagcctgca cgagcacatt gccaatctgg ccggcagccc
     4981 cgccattaag aagggcatcc tgcagacagt gaaggtggtg gacgagctcg tgaaagtgat
     5041 gggccggcac aagcccgaga acatcgtgat cgaaatggcc agagagaacc agaccaccca
     5101 gaagggacag aagaacagcc gcgagagaat gaagcggatc gaagagggca tcaaagagct
     5161 gggcagccag atcctgaaag aacaccccgt ggaaaacacc cagctgcaga acgagaagct
     5221 gtacctgtac tacctgcaga atgggcggga tatgtacgtg gaccaggaac tggacatcaa
     5281 ccggctgtcc gactacgatg tggacgctat cgtgcctcag agctttctga aggacgactc
     5341 catcgataac aaagtgctga ctcggagcga caagaaccgg ggcaagagcg acaacgtgcc
     5401 ctccgaagag gtcgtgaaga agatgaagaa ctactggcgc cagctgctga atgccaagct
     5461 gattacccag aggaagttcg acaatctgac caaggccgag agaggcggcc tgagcgaact
     5521 ggataaggcc ggcttcatca agagacagct ggtggaaacc cggcagatca caaagcacgt
     5581 ggcacagatc ctggactccc ggatgaacac taagtacgac gagaacgaca aactgatccg
     5641 ggaagtgaaa gtgatcaccc tgaagtccaa gctggtgtcc gatttccgga aggatttcca
     5701 gttttacaaa gtgcgcgaga tcaacaacta ccaccacgcc cacgacgcct acctgaacgc
     5761 cgtcgtggga accgccctga tcaaaaagta ccctaagctg gaaagcgagt tcgtgtacgg
     5821 cgactacaag gtgtacgacg tgcggaagat gatcgccaag agcgagcagg aaatcggcaa
     5881 ggctaccgcc aagtacttct tctacagcaa catcatgaac tttttcaaga ccgagattac
     5941 cctggccaac ggcgagatcc ggaagcggcc tctgatcgag acaaacggcg aaacaggcga
     6001 gatcgtgtgg gataagggcc gggactttgc caccgtgcgg aaagtgctgt ctatgcccca
     6061 agtgaatatc gtgaaaaaga ccgaggtgca gacaggcggc ttcagcaaag agtctatcct
     6121 gcccaagagg aacagcgaca agctgatcgc cagaaagaag gactgggacc ctaagaagta
     6181 cggcggcttc gacagcccca ccgtggccta ttctgtgctg gtggtggcca aagtggaaaa
     6241 gggcaagtcc aagaaactga agagtgtgaa agagctgctg gggatcacca tcatggaaag
     6301 aagcagcttc gagaagaatc ccatcgactt tctggaagcc aagggctaca aagaagtgaa
     6361 aaaggacctg atcatcaagc tgcctaagta ctccctgttc gagctggaaa acggccggaa
     6421 gagaatgctg gcctctgccg gcgaactgca gaagggaaac gaactggccc tgccctccaa
     6481 atatgtgaac ttcctgtacc tggccagcca ctatgagaag ctgaagggct cccccgagga
     6541 taatgagcag aaacagctgt ttgtggaaca gcacaaacac tacctggacg agatcatcga
     6601 gcagatcagc gagttctcca agagagtgat cctggccgac gctaatctgg acaaggtgct
     6661 gagcgcctac aacaagcaca gagacaagcc tatcagagag caggccgaga atatcatcca
     6721 cctgtttacc ctgaccaatc tgggagcccc tgccgccttc aagtactttg acaccaccat
     6781 cgaccggaag aggtacacca gcaccaaaga ggtgctggac gccaccctga tccaccagag
     6841 catcaccggc ctgtacgaga cacggatcga cctgtctcag ctgggaggcg acgcctatcc
     6901 ctatgacgtg cccgattatg ccagcctggg cagcggctcc cccaagaaaa aacgcaaggt
     6961 ggaagatcct aagaaaaagc ggaaagtgga cggcattggt agtgggagca acggcagcag
     7021 cggatccgtg agcaagggcg aggaggataa catggccatc atcaaggagt tcatgcgctt
     7081 caaggtgcac atggagggct ccgtgaacgg ccacgagttc gagatcgagg gcgagggcga
     7141 gggccgcccc tacgagggca cccagaccgc caagctgaag gtgaccaagg gtggccccct
     7201 gcccttcgcc tgggacatcc tgtcccctca gttcatgtac ggctccaagg cctacgtgaa
     7261 gcaccccgcc gacatccccg actacttgaa gctgtccttc cccgagggct tcaagtggga
     7321 gcgcgtgatg aacttcgagg acggcggcgt ggtgaccgtg acccaggact cctccctgca
     7381 ggacggcgag ttcatctaca aggtgaagct gcgcggcacc aacttcccct ccgacggccc
     7441 cgtaatgcag aagaagacca tgggctggga ggcctcctcc gagcggatgt accccgagga
     7501 cggcgccctg aagggcgaga tcaagcagag gctgaagctg aaggacggcg gccactacga
     7561 cgctgaggtc aagaccacct acaaggccaa gaagcccgtg cagctgcccg gcgcctacaa
     7621 cgtcaacatc aagttggaca tcacctccca caacgaggac tacaccatcg tggaacagta
     7681 cgaacgcgcc gagggccgcc actccaccgg cggcatggac gagctgtaca agggcggtgg
     7741 cggagggatg gatgctaagt cactaactgc ctggtcccgg acactggtga ccttcaagga
     7801 tgtatttgtg gacttcacca gggaggagtg gaagctgctg gacactgctc agcagatcgt
     7861 gtacagaaat gtgatgctgg agaactataa gaacctggtt tccttgggtt atcagcttac
     7921 taagccagat gtgatcctcc ggttggagaa gggagaagag ccctagtaag gccgcgactc
     7981 tagagtcgac ctgcaggcat gcaagcttga tatcaagctt atcgataatc aacctctgga
     8041 ttacaaaatt tgtgaaagat tgactggtat tcttaactat gttgctcctt ttacgctatg
     8101 tggatacgct gctttaatgc ctttgtatca tgctattgct tcccgtatgg ctttcatttt
     8161 ctcctccttg tataaatcct ggttgctgtc tctttatgag gagttgtggc ccgttgtcag
     8221 gcaacgtggc gtggtgtgca ctgtgtttgc tgacgcaacc cccactggtt ggggcattgc
     8281 caccacctgt cagctccttt ccgggacttt cgctttcccc ctccctattg ccacggcgga
     8341 actcatcgcc gcctgccttg cccgctgctg gacaggggct cggctgttgg gcactgacaa
     8401 ttccgtggtg ttgtcgggga aatcatcgtc ctttccttgg ctgctcgcct gtgttgccac
     8461 ctggattctg cgcgggacgt ccttctgcta cgtcccttcg gccctcaatc cagcggacct
     8521 tccttcccgc ggcctgctgc cggctctgcg gcctcttccg cgtcttcgcc ttcgccctca
     8581 gacgagtcgg atctcccttt gggccgcctc cccgcatcga taccgtcgac ctcgagggaa
     8641 ttaattcgag ctcggtacct ttaagaccaa tgacttacaa ggcagctgta gatcttagcc
     8701 actttttaaa agaaaagggg ggactggaag ggctaattca ctcccaacga agacaagatc
     8761 tgctttttgc ttgtactggg tctctctggt tagaccagat ctgagcctgg gagctctctg
     8821 gctaactagg gaacccactg cttaagcctc aataaagctt gccttgagtg cttcaagtag
     8881 tgtgtgcccg tctgttgtgt gactctggta actagagatc cctcagaccc ttttagtcag
     8941 tgtggaaaat ctctagcagc atctagaatt aattccgtgt attctatagt gtcacctaaa
     9001 tcgtatgtgt atgatacata aggttatgta ttaattgtag ccgcgttcta acgacaatat
     9061 gtacaagcct aattgtgtag catctggctt actgaagcag accctatcat ctctctcgta
     9121 aactgccgtc agagtcggtt tggttggacg aaccttctga gtttctggta acgccgtccc
     9181 gcacccggaa atggtcagcg aaccaatcag cagggtcatc gctagccaga tcctctacgc
     9241 cggacgcatc gtggccggca tcaccggcgc cacaggtgcg gttgctggcg cctatatcgc
     9301 cgacatcacc gatggggaag atcgggctcg ccacttcggg ctcatgagcg cttgtttcgg
     9361 cgtgggtatg gtggcaggcc ccgtggccgg gggactgttg ggcgccatct ccttgcatgc
     9421 accattcctt gcggcggcgg tgctcaacgg cctcaaccta ctactgggct gcttcctaat
     9481 gcaggagtcg cataagggag agcgtcgaat ggtgcactct cagtacaatc tgctctgatg
     9541 ccgcatagtt aagccagccc cgacacccgc caacacccgc tgacgcgccc tgacgggctt
     9601 gtctgctccc ggcatccgct tacagacaag ctgtgaccgt ctccgggagc tgcatgtgtc
     9661 agaggttttc accgtcatca ccgaaacgcg cgagacgaaa gggcctcgtg atacgcctat
     9721 ttttataggt taatgtcatg ataataatgg tttcttagac gtcaggtggc acttttcggg
     9781 gaaatgtgcg cggaacccct atttgtttat ttttctaaat acattcaaat atgtatccgc
     9841 tcatgagaca ataaccctga taaatgcttc aataatattg aaaaaggaag agtatgagta
     9901 ttcaacattt ccgtgtcgcc cttattccct tttttgcggc attttgcctt cctgtttttg
     9961 ctcacccaga aacgctggtg aaagtaaaag atgctgaaga tcagttgggt gcacgagtgg
    10021 gttacatcga actggatctc aacagcggta agatccttga gagttttcgc cccgaagaac
    10081 gttttccaat gatgagcact tttaaagttc tgctatgtgg cgcggtatta tcccgtattg
    10141 acgccgggca agagcaactc ggtcgccgca tacactattc tcagaatgac ttggttgagt
    10201 actcaccagt cacagaaaag catcttacgg atggcatgac agtaagagaa ttatgcagtg
    10261 ctgccataac catgagtgat aacactgcgg ccaacttact tctgacaacg atcggaggac
    10321 cgaaggagct aaccgctttt ttgcacaaca tgggggatca tgtaactcgc cttgatcgtt
    10381 gggaaccgga gctgaatgaa gccataccaa acgacgagcg tgacaccacg atgcctgtag
    10441 caatggcaac aacgttgcgc aaactattaa ctggcgaact acttactcta gcttcccggc
    10501 aacaattaat agactggatg gaggcggata aagttgcagg accacttctg cgctcggccc
    10561 ttccggctgg ctggtttatt gctgataaat ctggagccgg tgagcgtggg tctcgcggta
    10621 tcattgcagc actggggcca gatggtaagc cctcccgtat cgtagttatc tacacgacgg
    10681 ggagtcaggc aactatggat gaacgaaata gacagatcgc tgagataggt gcctcactga
    10741 ttaagcattg gtaactgtca gaccaagttt actcatatat actttagatt gatttaaaac
    10801 ttcattttta atttaaaagg atctaggtga agatcctttt tgataatctc atgaccaaaa
    10861 tcccttaacg tgagttttcg ttccactgag cgtcagaccc cgtagaaaag atcaaaggat
    10921 cttcttgaga tccttttttt ctgcgcgtaa tctgctgctt gcaaacaaaa aaaccaccgc
    10981 taccagcggt ggtttgtttg ccggatcaag agctaccaac tctttttccg aaggtaactg
    11041 gcttcagcag agcgcagata ccaaatactg ttcttctagt gtagccgtag ttaggccacc
    11101 acttcaagaa ctctgtagca ccgcctacat acctcgctct gctaatcctg ttaccagtgg
    11161 ctgctgccag tggcgataag tcgtgtctta ccgggttgga ctcaagacga tagttaccgg
    11221 ataaggcgca gcggtcgggc tgaacggggg gttcgtgcac acagcccagc ttggagcgaa
    11281 cgacctacac cgaactgaga tacctacagc gtgagctatg agaaagcgcc acgcttcccg
    11341 aagggagaaa ggcggacagg tatccggtaa gcggcagggt cggaacagga gagcgcacga
    11401 gggagcttcc agggggaaac gcctggtatc tttatagtcc tgtcgggttt cgccacctct
    11461 gacttgagcg tcgatttttg tgatgctcgt caggggggcg gagcctatgg aaaaacgcca
    11521 gcaacgcggc ctttttacgg ttcctggcct tttgctggcc ttttgctcac atgttctttc
    11581 ctgcgttatc ccctgattct gtggataacc gtattaccgc ctttgagtga gctgataccg
    11641 ctcgccgcag ccgaacgacc gagcgcagcg agtcagtgag cgaggaagcg gaagagcgcc
    11701 caatacgcaa accgcctctc cccgcgcgtt ggccgattca ttaatgcagc tgtggaatgt
    11761 gtgtcagtta gggtgtggaa agtccccagg ctccccagca ggcagaagta tgcaaagcat
    11821 gcatctcaat tagtcagcaa ccaggtgtgg aaagtcccca ggctccccag caggcagaag
    11881 tatgcaaagc atgcatctca attagtcagc aaccatagtc ccgcccctaa ctccgcccat
    11941 cccgccccta actccgccca gttccgccca ttctccgccc catggctgac taattttttt
    12001 tatttatgca gaggccgagg ccgcctcggc ctctgagcta ttccagaagt agtgaggagg
    12061 cttttttgga ggcctaggct tttgcaaaaa gcttggacac aagacaggct tgcgagatat
    12121 gtttgagaat accactttat cccgcgtcag ggagaggcag tgcgtaaaaa gacgcggact
    12181 catgtgaaat actggttttt agtgcgccag atctctataa tctcgcgcaa cctattttcc
    12241 cctcgaacac tttttaagcc gtagataaac aggctgggac acttcacatg agcgaaaaat
    12301 acatcgtcac ctgggacatg ttgcagatcc atgcacgtaa actcgcaagc cgactgatgc
    12361 cttctgaaca atggaaaggc attattgccg taagccgtgg cggtctgtac cgggtgcgtt
    12421 actggcgcgt gaactgggta ttcgtcatgt cgataccgtt tgtatttcca gctacgatca
    12481 cgacaaccag cgcgagctta aagtgctgaa acgcgcagaa ggcgatggcg aaggcttcat
    12541 cgttattgat gacctggtgg ataccggtgg tactgcggtt gcgattcgtg aaatgtatcc
    12601 aaaagcgcac tttgtcacca tcttcgcaaa accggctggt cgtccgctgg ttgatgacta
    12661 tgttgttgat atcccgcaag atacctggat tgaacagccg tgggatatgg gcgtcgtatt
    12721 cgtcccgcca atctccggtc gctaatcttt tcaacgcctg gcactgccgg gcgttgttct
    12781 ttttaacttc aggcgggtta caatagtttc cagtaagtat tctggaggct gcatccatga
    12841 cacaggcaaa cctgagcgaa accctgttca aaccccgctt taaacatcct gaaacctcga
    12901 cgctagtccg ccgctttaat cacggcgcac aaccgcctgt gcagtcggcc cttgatggta
    12961 aaaccatccc tcactggtat cgcatgatta accgtctgat gtggatctgg cgcggcattg
    13021 acccacgcga aatcctcgac gtccaggcac gtattgtgat gagcgatgcc gaacgtaccg
    13081 acgatgattt atacgatacg gtgattggct accgtggcgg caactggatt tatgagtggg
    13141 ccccggatct ttgtgaagga accttacttc tgtggtgtga cataattgga caaactacct
    13201 acagagattt aaagctctaa ggtaaatata aaatttttaa gtgtataatg tgttaaacta
    13261 ctgattctaa ttgtttgtgt attttagatt ccaacctatg gaactgatga atgggagcag
    13321 tggtggaatg cctttaatga ggaaaacctg ttttgctcag aagaaatgcc atctagtgat
    13381 gatgaggcta ctgctgactc tcaacattct actcctccaa aaaagaagag aaaggtagaa
    13441 gaccccaagg actttccttc agaattgcta agttttttga gtcatgctgt gtttagtaat
    13501 agaactcttg cttgctttgc tatttacacc acaaaggaaa aagctgcact gctatacaag
    13561 aaaattatgg aaaaatattc tgtaaccttt ataagtaggc ataacagtta taatcataac
    13621 atactgtttt ttcttactcc acacaggcat agagtgtctg ctattaataa ctatgctcaa
    13681 aaattgtgta cctttagctt tttaatttgt aaaggggtta ataaggaata tttgatgtat
    13741 agtgccttga ctagagatca taatcagcca taccacattt gtagaggttt tacttgcttt
    13801 aaaaaacctc ccacacctcc ccctgaacct gaaacataaa atgaatgcaa ttgttgttgt
    13861 taacttgttt attgcagctt ataatggtta caaataaagc aatagcatca caaatttcac
    13921 aaataaagca tttttttcac tgcattctag ttgtggtttg tccaaactca tcaatgtatc
    13981 ttatcatgtc tggatcaact ggataactca agctaaccaa aatcatccca aacttcccac
    14041 cccataccct attaccactg ccaattacct gtggtttcat ttactctaaa cctgtgattc
    14101 ctctgaatta ttttcatttt aaagaaattg tatttgttaa atatgtacta caaacttagt
    14161 agttggaagg gctaat
//
