LOCUS       sequence-374878-       16354 bp    DNA     circular SYN 14-APR-2025
DEFINITION  dCas9 with a C-term HA-2xNLS-XTEN80(linker)-KRAB(Kox1).
ACCESSION   .
VERSION     .
KEYWORDS    .
SOURCE      synthetic DNA construct
  ORGANISM  synthetic DNA construct
REFERENCE   1  (bases 1 to 16354)
  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 16354)
  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-EF1a-dCas9-XTEN80-KRAB(Kox1)-IRES-mCh...
FEATURES             Location/Qualifiers
     source          1..16354
                     /mol_type="other DNA"
                     /organism="synthetic DNA construct"
     promoter        complement(93..111)
                     /label=SP6 promoter
                     /note="promoter for bacteriophage SP6 RNA polymerase"
     primer_bind     complement(94..111)
                     /label=SP6
                     /note="SP6 promoter, forward primer"
     primer_bind     complement(401..420)
                     /label=pBRrevBam
                     /note="pBR322 vectors, tet region, downstream of BamHI, 
                     reverse primer"
     primer_bind     complement(634..653)
                     /label=pRS-marker
                     /note="pRS vectors, use to sequence yeast selectable 
                     marker"
     primer_bind     753..775
                     /label=pGEX 3'
                     /note="pGEX vectors, reverse primer"
     primer_bind     complement(813..831)
                     /label=pBRforEco
                     /note="pBR322 vectors, upsteam of EcoRI site, forward 
                     primer"
     promoter        899..1003
                     /gene="bla"
                     /label=AmpR promoter
     CDS             join(1004..1072,1073..1864)
                     /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(1222..1241)
                     /label=Amp-R
                     /note="Ampicillin resistance gene, reverse primer"
     rep_origin      2035..2623
                     /direction=RIGHT
                     /label=ori
                     /note="high-copy-number ColE1/pMB1/pBR322/pUC origin of 
                     replication"
     primer_bind     2524..2543
                     /label=pBR322ori-F
                     /note="pBR322 origin, forward primer"
     primer_bind     2777..2794
                     /label=L4440
                     /note="L4440 vector, forward primer"
     primer_bind     complement(2864..2884)
                     /label=pBABE 3'
                     /note="SV40 enhancer, reverse primer for pBABE vectors"
     promoter        2869..3198
                     /label=SV40 promoter
                     /note="SV40 enhancer and early promoter"
     rep_origin      3049..3184
                     /label=SV40 ori
                     /note="SV40 origin of replication"
     intron          4332..4397
                     /label=small t intron
                     /note="SV40 (simian virus 40) small t antigen intron"
     CDS             4527..4547
                     /codon_start=1
                     /product="nuclear localization signal of SV40 (simian virus
                     40) large T antigen"
                     /label=SV40 NLS
                     /translation="PKKKRKV"
     polyA_signal    4972..5106
                     /label=SV40 poly(A) signal
                     /note="SV40 polyadenylation signal"
     primer_bind     complement(5009..5028)
                     /label=SV40pA-R
                     /note="SV40 polyA, reverse primer"
     primer_bind     5063..5082
                     /label=EBV-rev
                     /note="SV40 polyA terminator, reverse primer"
     LTR             5274..5907
                     /label=3' LTR
                     /note="3' long terminal repeat (LTR) from HIV-1"
     misc_feature    5954..6079
                     /label=HIV-1 Psi
                     /note="packaging signal of human immunodeficiency virus 
                     type 1"
     misc_feature    6576..6809
                     /label=RRE
                     /note="The Rev response element (RRE) of HIV-1 allows for 
                     Rev-dependent mRNA export from the nucleus to the 
                     cytoplasm."
     CDS             6994..7038
                     /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    7323..7440
                     /label=cPPT/CTS
                     /note="central polypurine tract and central termination 
                     sequence of HIV-1"
     promoter        8172..9349
                     /label=EF-1-alpha promoter
                     /note="strong constitutive promoter for human elongation 
                     factor EF-1-alpha"
     intron          8403..9341
                     /label=EF-1-alpha intron A
                     /note="intron upstream of the start codon of human 
                     EF-1-alpha"
     primer_bind     9298..9318
                     /label=EF1a-F
                     /note="Human elongation factor-1a promoter, forward primer"
     regulatory      9399..9408
                     /label=Kozak sequence
                     /note="vertebrate consensus sequence for strong initiation 
                     of translation (Kozak, 1987)"
                     /regulatory_class="other"
     CDS             join(9405..9431,9432..9434,9435..11921,11922..11924,
                     11925..13508)
                     /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             9405..13508
                     /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             13512..13538
                     /codon_start=1
                     /product="HA (human influenza hemagglutinin) epitope tag"
                     /label=HA
                     /translation="YPYDVPDYA"
     CDS             13518..13544
                     /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             13557..13577
                     /codon_start=1
                     /product="nuclear localization signal of SV40 (simian virus
                     40) large T antigen"
                     /label=SV40 NLS
                     /translation="PKKKRKV"
     CDS             13584..13604
                     /codon_start=1
                     /product="nuclear localization signal of SV40 (simian virus
                     40) large T antigen"
                     /label=SV40 NLS
                     /translation="PKKKRKV"
     CDS             13914..14099
                     /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    14138..14705
                     /label=IRES
                     /note="internal ribosome entry site (IRES) of the 
                     encephalomyocarditis virus (EMCV)"
     misc_feature    14155..14707
                     /label=IRES
                     /note="internal ribosome entry site (IRES) of the 
                     encephalomyocarditis virus (EMCV)"
     primer_bind     complement(14299..14316)
                     /label=IRES reverse
                     /note="IRES internal ribosome entry site, reverse primer. 
                     Also called pCDH-rev"
     primer_bind     14526..14545
                     /label=IRES-F
                     /note="IRES internal ribosome entry site, forward primer"
     CDS             14723..15433
                     /codon_start=1
                     /product="monomeric derivative of DsRed fluorescent protein
                     (Shaner et al., 2004)"
                     /label=mCherry
                     /note="mammalian codon-optimized"
                     /translation="MVSKGEEDNMAIIKEFMRFKVHMEGSVNGHEFEIEGEGEGRPYEG
                     TQTAKLKVTKGGPLPFAWDILSPQFMYGSKAYVKHPADIPDYLKLSFPEGFKWERVMNF
                     EDGGVVTVTQDSSLQDGEFIYKVKLRGTNFPSDGPVMQKKTMGWEASSERMYPEDGALK
                     GEIKQRLKLKDGGHYDAEVKTTYKAKKPVQLPGAYNVNIKLDITSHNEDYTIVEQYERA
                     EGRHSTGGMDELYK"
     primer_bind     complement(14868..14886)
                     /label=mCherry-R
                     /note="mCherry, reverse primer"
     primer_bind     15136..15155
                     /label=mCherry-F
                     /note="mCherry, forward primer"
     misc_feature    15491..16079
                     /label=WPRE
                     /note="woodchuck hepatitis virus posttranscriptional 
                     regulatory element"
     primer_bind     complement(15544..15564)
                     /label=WPRE-R
                     /note="WPRE, reverse primer"
     CDS             complement(15962..15973)
                     /codon_start=1
                     /product="Factor Xa recognition and cleavage site"
                     /label=Factor Xa site
                     /translation="IEGR"
     primer_bind     complement(16082..16098)
                     /label=KS primer
                     /note="common sequencing primer, one of multiple similar 
                     variants"
     primer_bind     complement(16083..16099)
                     /label=pBluescriptKS
                     /note="For pBluescript vector"
     LTR             join(16189..16354,1..68)
                     /label=3' LTR (Delta-U3)
                     /note="self-inactivating 3' long terminal repeat (LTR) from
                     HIV-1"
ORIGIN
        1 tctgttgtgt gactctggta actagagatc cctcagaccc ttttagtcag tgtggaaaat
       61 ctctagcagc atctagaatt aattccgtgt attctatagt gtcacctaaa tcgtatgtgt
      121 atgatacata aggttatgta ttaattgtag ccgcgttcta acgacaatat gtacaagcct
      181 aattgtgtag catctggctt actgaagcag accctatcat ctctctcgta aactgccgtc
      241 agagtcggtt tggttggacg aaccttctga gtttctggta acgccgtccc gcacccggaa
      301 atggtcagcg aaccaatcag cagggtcatc gctagccaga tcctctacgc cggacgcatc
      361 gtggccggca tcaccggcgc cacaggtgcg gttgctggcg cctatatcgc cgacatcacc
      421 gatggggaag atcgggctcg ccacttcggg ctcatgagcg cttgtttcgg cgtgggtatg
      481 gtggcaggcc ccgtggccgg gggactgttg ggcgccatct ccttgcatgc accattcctt
      541 gcggcggcgg tgctcaacgg cctcaaccta ctactgggct gcttcctaat gcaggagtcg
      601 cataagggag agcgtcgaat ggtgcactct cagtacaatc tgctctgatg ccgcatagtt
      661 aagccagccc cgacacccgc caacacccgc tgacgcgccc tgacgggctt gtctgctccc
      721 ggcatccgct tacagacaag ctgtgaccgt ctccgggagc tgcatgtgtc agaggttttc
      781 accgtcatca ccgaaacgcg cgagacgaaa gggcctcgtg atacgcctat ttttataggt
      841 taatgtcatg ataataatgg tttcttagac gtcaggtggc acttttcggg gaaatgtgcg
      901 cggaacccct atttgtttat ttttctaaat acattcaaat atgtatccgc tcatgagaca
      961 ataaccctga taaatgcttc aataatattg aaaaaggaag agtatgagta ttcaacattt
     1021 ccgtgtcgcc cttattccct tttttgcggc attttgcctt cctgtttttg ctcacccaga
     1081 aacgctggtg aaagtaaaag atgctgaaga tcagttgggt gcacgagtgg gttacatcga
     1141 actggatctc aacagcggta agatccttga gagttttcgc cccgaagaac gttttccaat
     1201 gatgagcact tttaaagttc tgctatgtgg cgcggtatta tcccgtattg acgccgggca
     1261 agagcaactc ggtcgccgca tacactattc tcagaatgac ttggttgagt actcaccagt
     1321 cacagaaaag catcttacgg atggcatgac agtaagagaa ttatgcagtg ctgccataac
     1381 catgagtgat aacactgcgg ccaacttact tctgacaacg atcggaggac cgaaggagct
     1441 aaccgctttt ttgcacaaca tgggggatca tgtaactcgc cttgatcgtt gggaaccgga
     1501 gctgaatgaa gccataccaa acgacgagcg tgacaccacg atgcctgtag caatggcaac
     1561 aacgttgcgc aaactattaa ctggcgaact acttactcta gcttcccggc aacaattaat
     1621 agactggatg gaggcggata aagttgcagg accacttctg cgctcggccc ttccggctgg
     1681 ctggtttatt gctgataaat ctggagccgg tgagcgtggg tctcgcggta tcattgcagc
     1741 actggggcca gatggtaagc cctcccgtat cgtagttatc tacacgacgg ggagtcaggc
     1801 aactatggat gaacgaaata gacagatcgc tgagataggt gcctcactga ttaagcattg
     1861 gtaactgtca gaccaagttt actcatatat actttagatt gatttaaaac ttcattttta
     1921 atttaaaagg atctaggtga agatcctttt tgataatctc atgaccaaaa tcccttaacg
     1981 tgagttttcg ttccactgag cgtcagaccc cgtagaaaag atcaaaggat cttcttgaga
     2041 tccttttttt ctgcgcgtaa tctgctgctt gcaaacaaaa aaaccaccgc taccagcggt
     2101 ggtttgtttg ccggatcaag agctaccaac tctttttccg aaggtaactg gcttcagcag
     2161 agcgcagata ccaaatactg ttcttctagt gtagccgtag ttaggccacc acttcaagaa
     2221 ctctgtagca ccgcctacat acctcgctct gctaatcctg ttaccagtgg ctgctgccag
     2281 tggcgataag tcgtgtctta ccgggttgga ctcaagacga tagttaccgg ataaggcgca
     2341 gcggtcgggc tgaacggggg gttcgtgcac acagcccagc ttggagcgaa cgacctacac
     2401 cgaactgaga tacctacagc gtgagctatg agaaagcgcc acgcttcccg aagggagaaa
     2461 ggcggacagg tatccggtaa gcggcagggt cggaacagga gagcgcacga gggagcttcc
     2521 agggggaaac gcctggtatc tttatagtcc tgtcgggttt cgccacctct gacttgagcg
     2581 tcgatttttg tgatgctcgt caggggggcg gagcctatgg aaaaacgcca gcaacgcggc
     2641 ctttttacgg ttcctggcct tttgctggcc ttttgctcac atgttctttc ctgcgttatc
     2701 ccctgattct gtggataacc gtattaccgc ctttgagtga gctgataccg ctcgccgcag
     2761 ccgaacgacc gagcgcagcg agtcagtgag cgaggaagcg gaagagcgcc caatacgcaa
     2821 accgcctctc cccgcgcgtt ggccgattca ttaatgcagc tgtggaatgt gtgtcagtta
     2881 gggtgtggaa agtccccagg ctccccagca ggcagaagta tgcaaagcat gcatctcaat
     2941 tagtcagcaa ccaggtgtgg aaagtcccca ggctccccag caggcagaag tatgcaaagc
     3001 atgcatctca attagtcagc aaccatagtc ccgcccctaa ctccgcccat cccgccccta
     3061 actccgccca gttccgccca ttctccgccc catggctgac taattttttt tatttatgca
     3121 gaggccgagg ccgcctcggc ctctgagcta ttccagaagt agtgaggagg cttttttgga
     3181 ggcctaggct tttgcaaaaa gcttggacac aagacaggct tgcgagatat gtttgagaat
     3241 accactttat cccgcgtcag ggagaggcag tgcgtaaaaa gacgcggact catgtgaaat
     3301 actggttttt agtgcgccag atctctataa tctcgcgcaa cctattttcc cctcgaacac
     3361 tttttaagcc gtagataaac aggctgggac acttcacatg agcgaaaaat acatcgtcac
     3421 ctgggacatg ttgcagatcc atgcacgtaa actcgcaagc cgactgatgc cttctgaaca
     3481 atggaaaggc attattgccg taagccgtgg cggtctgtac cgggtgcgtt actggcgcgt
     3541 gaactgggta ttcgtcatgt cgataccgtt tgtatttcca gctacgatca cgacaaccag
     3601 cgcgagctta aagtgctgaa acgcgcagaa ggcgatggcg aaggcttcat cgttattgat
     3661 gacctggtgg ataccggtgg tactgcggtt gcgattcgtg aaatgtatcc aaaagcgcac
     3721 tttgtcacca tcttcgcaaa accggctggt cgtccgctgg ttgatgacta tgttgttgat
     3781 atcccgcaag atacctggat tgaacagccg tgggatatgg gcgtcgtatt cgtcccgcca
     3841 atctccggtc gctaatcttt tcaacgcctg gcactgccgg gcgttgttct ttttaacttc
     3901 aggcgggtta caatagtttc cagtaagtat tctggaggct gcatccatga cacaggcaaa
     3961 cctgagcgaa accctgttca aaccccgctt taaacatcct gaaacctcga cgctagtccg
     4021 ccgctttaat cacggcgcac aaccgcctgt gcagtcggcc cttgatggta aaaccatccc
     4081 tcactggtat cgcatgatta accgtctgat gtggatctgg cgcggcattg acccacgcga
     4141 aatcctcgac gtccaggcac gtattgtgat gagcgatgcc gaacgtaccg acgatgattt
     4201 atacgatacg gtgattggct accgtggcgg caactggatt tatgagtggg ccccggatct
     4261 ttgtgaagga accttacttc tgtggtgtga cataattgga caaactacct acagagattt
     4321 aaagctctaa ggtaaatata aaatttttaa gtgtataatg tgttaaacta ctgattctaa
     4381 ttgtttgtgt attttagatt ccaacctatg gaactgatga atgggagcag tggtggaatg
     4441 cctttaatga ggaaaacctg ttttgctcag aagaaatgcc atctagtgat gatgaggcta
     4501 ctgctgactc tcaacattct actcctccaa aaaagaagag aaaggtagaa gaccccaagg
     4561 actttccttc agaattgcta agttttttga gtcatgctgt gtttagtaat agaactcttg
     4621 cttgctttgc tatttacacc acaaaggaaa aagctgcact gctatacaag aaaattatgg
     4681 aaaaatattc tgtaaccttt ataagtaggc ataacagtta taatcataac atactgtttt
     4741 ttcttactcc acacaggcat agagtgtctg ctattaataa ctatgctcaa aaattgtgta
     4801 cctttagctt tttaatttgt aaaggggtta ataaggaata tttgatgtat agtgccttga
     4861 ctagagatca taatcagcca taccacattt gtagaggttt tacttgcttt aaaaaacctc
     4921 ccacacctcc ccctgaacct gaaacataaa atgaatgcaa ttgttgttgt taacttgttt
     4981 attgcagctt ataatggtta caaataaagc aatagcatca caaatttcac aaataaagca
     5041 tttttttcac tgcattctag ttgtggtttg tccaaactca tcaatgtatc ttatcatgtc
     5101 tggatcaact ggataactca agctaaccaa aatcatccca aacttcccac cccataccct
     5161 attaccactg ccaattacct gtggtttcat ttactctaaa cctgtgattc ctctgaatta
     5221 ttttcatttt aaagaaattg tatttgttaa atatgtacta caaacttagt agttggaagg
     5281 gctaattcac tcccaaagaa gacaagatat ccttgatctg tggatctacc acacacaagg
     5341 ctacttccct gattagcaga actacacacc agggccaggg gtcagatatc cactgacctt
     5401 tggatggtgc tacaagctag taccagttga gccagataag gtagaagagg ccaataaagg
     5461 agagaacacc agcttgttac accctgtgag cctgcatggg atggatgacc cggagagaga
     5521 agtgttagag tggaggtttg acagccgcct agcatttcat cacgtggccc gagagctgca
     5581 tccggagtac ttcaagaact gctgatatcg agcttgctac aagggacttt ccgctgggga
     5641 ctttccaggg aggcgtggcc tgggcgggac tggggagtgg cgagccctca gatcctgcat
     5701 ataagcagct gctttttgcc tgtactgggt ctctctggtt agaccagatc tgagcctggg
     5761 agctctctgg ctaactaggg aacccactgc ttaagcctca ataaagcttg ccttgagtgc
     5821 ttcaagtagt gtgtgcccgt ctgttgtgtg actctggtaa ctagagatcc ctcagaccct
     5881 tttagtcagt gtggaaaatc tctagcagtg gcgcccgaac agggacttga aagcgaaagg
     5941 gaaaccagag gagctctctc gacgcaggac tcggcttgct gaagcgcgca cggcaagagg
     6001 cgaggggcgg cgactggtga gtacgccaaa aattttgact agcggaggct agaaggagag
     6061 agatgggtgc gagagcgtca gtattaagcg ggggagaatt agatcgcgat gggaaaaaat
     6121 tcggttaagg ccagggggaa agaaaaaata taaattaaaa catatagtat gggcaagcag
     6181 ggagctagaa cgattcgcag ttaatcctgg cctgttagaa acatcagaag gctgtagaca
     6241 aatactggga cagctacaac catcccttca gacaggatca gaagaactta gatcattata
     6301 taatacagta gcaaccctct attgtgtgca tcaaaggata gagataaaag acaccaagga
     6361 agctttagac aagatagagg aagagcaaaa caaaagtaag accaccgcac agcaagcggc
     6421 cggccgctga tcttcagacc tggaggagga gatatgaggg acaattggag aagtgaatta
     6481 tataaatata aagtagtaaa aattgaacca ttaggagtag cacccaccaa ggcaaagaga
     6541 agagtggtgc agagagaaaa aagagcagtg ggaataggag ctttgttcct tgggttcttg
     6601 ggagcagcag gaagcactat gggcgcagcg tcaatgacgc tgacggtaca ggccagacaa
     6661 ttattgtctg gtatagtgca gcagcagaac aatttgctga gggctattga ggcgcaacag
     6721 catctgttgc aactcacagt ctggggcatc aagcagctcc aggcaagaat cctggctgtg
     6781 gaaagatacc taaaggatca acagctcctg gggatttggg gttgctctgg aaaactcatt
     6841 tgcaccactg ctgtgccttg gaatgctagt tggagtaata aatctctgga acagatttgg
     6901 aatcacacga cctggatgga gtgggacaga gaaattaaca attacacaag cttaatacac
     6961 tccttaattg aagaatcgca aaaccagcaa gaaaagaatg aacaagaatt attggaatta
     7021 gataaatggg caagtttgtg gaattggttt aacataacaa attggctgtg gtatataaaa
     7081 ttattcataa tgatagtagg aggcttggta ggtttaagaa tagtttttgc tgtactttct
     7141 atagtgaata gagttaggca gggatattca ccattatcgt ttcagaccca cctcccaacc
     7201 ccgaggggac ccgacaggcc cgaaggaata gaagaagaag gtggagagag agacagagac
     7261 agatccattc gattagtgaa cggatctcga cggtatcgcc aaatggcagt attcatccac
     7321 aattttaaaa gaaaaggggg gattgggggg tacagtgcag gggaaagaat agtagacata
     7381 atagcaacag acatacaaac taaagaatta caaaaacaaa ttacaaaaat tcaaaatttt
     7441 cgggtttatt acagggacag cagagatcca gtttggatcg ataagcttga tatcgaattc
     7501 gggaggtggt ccctgcagtt acgccaatga taacccccgc cagaaaaatc ttagtagcct
     7561 tccctttttg ttttccgtgc cccaactcgg cggattgact cggccccttc cggaaacacc
     7621 cgaatcaact tctagtcaaa ttattgttca cgccgcaatg acccacccct ggcccgcgtc
     7681 tgtggaactg acccctggtg tacaggagag ttcgctgctg aaagtggtcc caaaggggta
     7741 ctagttttta agctcccaac tccccctccc ccagcgtctg gaggattcca caccctcgca
     7801 ccgcaggggc gaggaagtgg gcggagtccg gttttggcgc cagccgctga ggctgccaag
     7861 cagaaaagcc accgctgagg agactccggt cactgtcctc gccccgcctc ccccttccct
     7921 ccccttgggg accaccgggc gccacgccgc gaacggtaag tgccgcggtc gtcggcgcct
     7981 ccgccctccc cctagggccc caattcccag cgggcgcggc gcgcggcccc tccccccgcc
     8041 gggcgcgcgc ccgctgcccc gcccttcgtg gccgcccggc gtgggcggtg ccacccctcc
     8101 ccccggcggc cccgcgcgca gctcccggct ccctccccct tcggatgtgg cttgagctgt
     8161 aggcgcggag gggctccggt gcccgtcagt gggcagagcg cacatcgccc acagtccccg
     8221 agaagttggg gggaggggtc ggcaattgaa ccggtgccta gagaaggtgg cgcggggtaa
     8281 actgggaaag tgatgtcgtg tactggctcc gcctttttcc cgagggtggg ggagaaccgt
     8341 atataagtgc agtagtcgcc gtgaacgttc tttttcgcaa cgggtttgcc gccagaacac
     8401 aggtaagtgc cgtgtgtggt tcccgcgggc ctggcctctt tacgggttat ggcccttgcg
     8461 tgccttgaat tacttccacc tggctgcagt acgtgattct tgatcccgag cttcgggttg
     8521 gaagtgggtg ggagagttcg aggccttgcg cttaaggagc cccttcgcct cgtgcttgag
     8581 ttgaggcctg gcctgggcgc tggggccgcc gcgtgcgaat ctggtggcac cttcgcgcct
     8641 gtctcgctgc tttcgataag tctctagcca tttaaaattt ttgatgacct gctgcgacgc
     8701 tttttttctg gcaagatagt cttgtaaatg cgggccaaga tctgcacact ggtatttcgg
     8761 tttttggggc cgcgggcggc gacggggccc gtgcgtccca gcgcacatgt tcggcgaggc
     8821 ggggcctgcg agcgcggcca ccgagaatcg gacgggggta gtctcaagct ggccggcctg
     8881 ctctggtgcc tggcctcgcg ccgccgtgta tcgccccgcc ctgggcggca aggctggccc
     8941 ggtcggcacc agttgcgtga gcggaaagat ggccgcttcc cggccctgct gcagggagct
     9001 caaaatggag gacgcggcgc tcgggagagc gggcgggtga gtcacccaca caaaggaaaa
     9061 gggcctttcc gtcctcagcc gtcgcttcat gtgactccac ggagtaccgg gcgccgtcca
     9121 ggcacctcga ttagttctcg agcttttgga gtacgtcgtc tttaggttgg ggggaggggt
     9181 tttatgcgat ggagtttccc cacactgagt gggtggagac tgaagttagg ccagcttggc
     9241 acttgatgta attctccttg gaatttgccc tttttgagtt tggatcttgg ttcattctca
     9301 agcctcagac agtggttcaa agtttttttc ttccatttca ggtgtcgtgg aattctcacg
     9361 cgtctgcagg atatcaagct tgcggtaccg cgggcccggc caccatggac aagaagtaca
     9421 gcatcggcct ggccatcggc accaactctg tgggctgggc cgtgatcacc gacgagtaca
     9481 aggtgcccag caagaaattc aaggtgctgg gcaacaccga ccggcacagc atcaagaaga
     9541 acctgatcgg cgccctgctg ttcgacagcg gagaaacagc cgaggccacc cggctgaaga
     9601 gaaccgccag aagaagatac accagacgga agaaccggat ctgctatctg caagagatct
     9661 tcagcaacga gatggccaag gtggacgaca gcttcttcca cagactggaa gagtccttcc
     9721 tggtggaaga ggataagaag cacgagcggc accccatctt cggcaacatc gtggacgagg
     9781 tggcctacca cgagaagtac cccaccatct accacctgag aaagaaactg gtggacagca
     9841 ccgacaaggc cgacctgcgg ctgatctatc tggccctggc ccacatgatc aagttccggg
     9901 gccacttcct gatcgagggc gacctgaacc ccgacaacag cgacgtggac aagctgttca
     9961 tccagctggt gcagacctac aaccagctgt tcgaggaaaa ccccatcaac gccagcggcg
    10021 tggacgccaa ggccatcctg tctgccagac tgagcaagag cagacggctg gaaaatctga
    10081 tcgcccagct gcccggcgag aagaagaatg gcctgttcgg caacctgatt gccctgagcc
    10141 tgggcctgac ccccaacttc aagagcaact tcgacctggc cgaggatgcc aaactgcagc
    10201 tgagcaagga cacctacgac gacgacctgg acaacctgct ggcccagatc ggcgaccagt
    10261 acgccgacct gtttctggcc gccaagaacc tgtccgacgc catcctgctg agcgacatcc
    10321 tgagagtgaa caccgagatc accaaggccc ccctgagcgc ctctatgatc aagagatacg
    10381 acgagcacca ccaggacctg accctgctga aagctctcgt gcggcagcag ctgcctgaga
    10441 agtacaaaga gattttcttc gaccagagca agaacggcta cgccggctac atcgatggcg
    10501 gagccagcca ggaagagttc tacaagttca tcaagcccat cctggaaaag atggacggca
    10561 ccgaggaact gctcgtgaag ctgaacagag aggacctgct gcggaagcag cggaccttcg
    10621 acaacggcag catcccccac cagatccacc tgggagagct gcacgccatt ctgcggcggc
    10681 aggaagattt ttacccattc ctgaaggaca accgggaaaa gatcgagaag atcctgacct
    10741 tccgcatccc ctactacgtg ggccctctgg ccaggggaaa cagcagattc gcctggatga
    10801 ccagaaagag cgaggaaacc atcaccccct ggaacttcga ggaagtggtg gacaagggcg
    10861 ccagcgccca gagcttcatc gagcggatga ccaacttcga taagaacctg cccaacgaga
    10921 aggtgctgcc caagcacagc ctgctgtacg agtacttcac cgtgtacaac gagctgacca
    10981 aagtgaaata cgtgaccgag ggaatgagaa agcccgcctt cctgagcggc gagcagaaaa
    11041 aagccatcgt ggacctgctg ttcaagacca accggaaagt gaccgtgaag cagctgaaag
    11101 aggactactt caagaaaatc gagtgcttcg actccgtgga aatctccggc gtggaagatc
    11161 ggttcaacgc ctccctgggc acataccacg atctgctgaa aattatcaag gacaaggact
    11221 tcctggacaa tgaggaaaac gaggacattc tggaagatat cgtgctgacc ctgacactgt
    11281 ttgaggacag agagatgatc gaggaacggc tgaaaaccta tgcccacctg ttcgacgaca
    11341 aagtgatgaa gcagctgaag cggcggagat acaccggctg gggcaggctg agccggaagc
    11401 tgatcaacgg catccgggac aagcagtccg gcaagacaat cctggatttc ctgaagtccg
    11461 acggcttcgc caacagaaac ttcatgcagc tgatccacga cgacagcctg acctttaaag
    11521 aggacatcca gaaagcccag gtgtccggcc agggcgatag cctgcacgag cacattgcca
    11581 atctggccgg cagccccgcc attaagaagg gcatcctgca gacagtgaag gtggtggacg
    11641 agctcgtgaa agtgatgggc cggcacaagc ccgagaacat cgtgatcgaa atggccagag
    11701 agaaccagac cacccagaag ggacagaaga acagccgcga gagaatgaag cggatcgaag
    11761 agggcatcaa agagctgggc agccagatcc tgaaagaaca ccccgtggaa aacacccagc
    11821 tgcagaacga gaagctgtac ctgtactacc tgcagaatgg gcgggatatg tacgtggacc
    11881 aggaactgga catcaaccgg ctgtccgact acgatgtgga cgctatcgtg cctcagagct
    11941 ttctgaagga cgactccatc gataacaaag tgctgactcg gagcgacaag aaccggggca
    12001 agagcgacaa cgtgccctcc gaagaggtcg tgaagaagat gaagaactac tggcgccagc
    12061 tgctgaatgc caagctgatt acccagagga agttcgacaa tctgaccaag gccgagagag
    12121 gcggcctgag cgaactggat aaggccggct tcatcaagag acagctggtg gaaacccggc
    12181 agatcacaaa gcacgtggca cagatcctgg actcccggat gaacactaag tacgacgaga
    12241 acgacaaact gatccgggaa gtgaaagtga tcaccctgaa gtccaagctg gtgtccgatt
    12301 tccggaagga tttccagttt tacaaagtgc gcgagatcaa caactaccac cacgcccacg
    12361 acgcctacct gaacgccgtc gtgggaaccg ccctgatcaa aaagtaccct aagctggaaa
    12421 gcgagttcgt gtacggcgac tacaaggtgt acgacgtgcg gaagatgatc gccaagagcg
    12481 agcaggaaat cggcaaggct accgccaagt acttcttcta cagcaacatc atgaactttt
    12541 tcaagaccga gattaccctg gccaacggcg agatccggaa gcggcctctg atcgagacaa
    12601 acggcgaaac aggcgagatc gtgtgggata agggccggga ctttgccacc gtgcggaaag
    12661 tgctgtctat gccccaagtg aatatcgtga aaaagaccga ggtgcagaca ggcggcttca
    12721 gcaaagagtc tatcctgccc aagaggaaca gcgacaagct gatcgccaga aagaaggact
    12781 gggaccctaa gaagtacggc ggcttcgaca gccccaccgt ggcctattct gtgctggtgg
    12841 tggccaaagt ggaaaagggc aagtccaaga aactgaagag tgtgaaagag ctgctgggga
    12901 tcaccatcat ggaaagaagc agcttcgaga agaatcccat cgactttctg gaagccaagg
    12961 gctacaaaga agtgaaaaag gacctgatca tcaagctgcc taagtactcc ctgttcgagc
    13021 tggaaaacgg ccggaagaga atgctggcct ctgccggcga actgcagaag ggaaacgaac
    13081 tggccctgcc ctccaaatat gtgaacttcc tgtacctggc cagccactat gagaagctga
    13141 agggctcccc cgaggataat gagcagaaac agctgtttgt ggaacagcac aaacactacc
    13201 tggacgagat catcgagcag atcagcgagt tctccaagag agtgatcctg gccgacgcta
    13261 atctggacaa ggtgctgagc gcctacaaca agcacagaga caagcctatc agagagcagg
    13321 ccgagaatat catccacctg tttaccctga ccaatctggg agcccctgcc gccttcaagt
    13381 actttgacac caccatcgac cggaagaggt acaccagcac caaagaggtg ctggacgcca
    13441 ccctgatcca ccagagcatc accggcctgt acgagacacg gatcgacctg tctcagctgg
    13501 gaggcgacgc ctatccctat gacgtgcccg attatgccag cctgggcagc ggctccccca
    13561 agaaaaaacg caaggtggaa gatcctaaga aaaagcggaa agtggacggc attggtagtg
    13621 ggagcaacgg cagcagcgga tccggagggc cgagctctgg cgcaccccca ccaagtggag
    13681 ggtctcctgc cgggtcccca acatctactg aagaaggcac cagcgaatcc gcaacgcccg
    13741 agtcaggccc tggtacctcc acagaaccat ctgaaggtag tgcgcctggt tccccagctg
    13801 gaagccctac ttccaccgaa gaaggcacgt caaccgaacc aagtgaagga tctgcccctg
    13861 ggaccagcac tgaaccatct gagatggatg ctaagtcact aactgcctgg tcccggacac
    13921 tggtgacctt caaggatgta tttgtggact tcaccaggga ggagtggaag ctgctggaca
    13981 ctgctcagca gatcgtgtac agaaatgtga tgctggagaa ctataagaac ctggtttcct
    14041 tgggttatca gcttactaag ccagatgtga tcctccggtt ggagaaggga gaagagccct
    14101 agtaagcggc cgcttagatc tctcgaggtt aacgaattcc gccccccccc cctaacgtta
    14161 ctggccgaag ccgcttggaa taaggccggt gtgcgtttgt ctatatgtta ttttccacca
    14221 tattgccgtc ttttggcaat gtgagggccc ggaaacctgg ccctgtcttc ttgacgagca
    14281 ttcctagggg tctttcccct ctcgccaaag gaatgcaagg tctgttgaat gtcgtgaagg
    14341 aagcagttcc tctggaagct tcttgaagac aaacaacgtc tgtagcgacc ctttgcaggc
    14401 agcggaaccc cccacctggc gacaggtgcc tctgcggcca aaagccacgt gtataagata
    14461 cacctgcaaa ggcggcacaa ccccagtgcc acgttgtgag ttggatagtt gtggaaagag
    14521 tcaaatggct ctcctcaagc gtattcaaca aggggctgaa ggatgcccag aaggtacccc
    14581 attgtatggg atctgatctg gggcctcggt gcacatgctt tacatgtgtt tagtcgaggt
    14641 taaaaaaacg tctaggcccc ccgaaccacg gggacgtggt tttcctttga aaaacacgat
    14701 gataatatgg ccacaaccat ggatggtgag caagggcgag gaggataaca tggccatcat
    14761 caaggagttc atgcgcttca aggtgcacat ggagggctcc gtgaacggcc acgagttcga
    14821 gatcgagggc gagggcgagg gccgccccta cgagggcacc cagaccgcca agctgaaggt
    14881 gaccaagggc ggccccctgc ccttcgcctg ggacatcctg tcccctcagt tcatgtacgg
    14941 ctccaaggcc tacgtgaagc accccgccga catccccgac tacttgaagc tgtccttccc
    15001 cgagggcttc aagtgggagc gcgtgatgaa cttcgaggac ggcggcgtgg tgaccgtgac
    15061 ccaggactcc tccctgcagg acggcgagtt catctacaag gtgaagctgc gcggcaccaa
    15121 cttcccctcc gacggccccg taatgcagaa gaagaccatg ggctgggagg cctcctccga
    15181 gcggatgtac cccgaggacg gcgccctgaa gggcgagatc aagcagaggc tgaagctgaa
    15241 ggacggcggc cactacgacg ctgaggtcaa gaccacctac aaggccaaga agcccgtgca
    15301 gctgcccggc gcctacaacg tcaacatcaa gttggacatc acctcccaca acgaggacta
    15361 caccatcgtg gaacagtacg aacgcgccga gggccgccac tccaccggcg gcatggacga
    15421 gctgtacaag taaggccgcg actctagagt cgacctgcag gcatgcaagc ttgatatcaa
    15481 gcttatcgat aatcaacctc tggattacaa aatttgtgaa agattgactg gtattcttaa
    15541 ctatgttgct ccttttacgc tatgtggata cgctgcttta atgcctttgt atcatgctat
    15601 tgcttcccgt atggctttca ttttctcctc cttgtataaa tcctggttgc tgtctcttta
    15661 tgaggagttg tggcccgttg tcaggcaacg tggcgtggtg tgcactgtgt ttgctgacgc
    15721 aacccccact ggttggggca ttgccaccac ctgtcagctc ctttccggga ctttcgcttt
    15781 ccccctccct attgccacgg cggaactcat cgccgcctgc cttgcccgct gctggacagg
    15841 ggctcggctg ttgggcactg acaattccgt ggtgttgtcg gggaaatcat cgtcctttcc
    15901 ttggctgctc gcctgtgttg ccacctggat tctgcgcggg acgtccttct gctacgtccc
    15961 ttcggccctc aatccagcgg accttccttc ccgcggcctg ctgccggctc tgcggcctct
    16021 tccgcgtctt cgccttcgcc ctcagacgag tcggatctcc ctttgggccg cctccccgca
    16081 tcgataccgt cgacctcgag ggaattaatt cgagctcggt acctttaaga ccaatgactt
    16141 acaaggcagc tgtagatctt agccactttt taaaagaaaa ggggggactg gaagggctaa
    16201 ttcactccca acgaagacaa gatctgcttt ttgcttgtac tgggtctctc tggttagacc
    16261 agatctgagc ctgggagctc tctggctaac tagggaaccc actgcttaag cctcaataaa
    16321 gcttgccttg agtgcttcaa gtagtgtgtg cccg
//
