LOCUS       sequence-374881-       15707 bp    DNA     circular SYN 15-DEC-2025
DEFINITION  dCas9 with an N-term Zim3 KRAB-NLS fusion, a C-term HA-2xNLS.
ACCESSION   .
VERSION     .
KEYWORDS    .
SOURCE      synthetic DNA construct
  ORGANISM  synthetic DNA construct
REFERENCE   1  (bases 1 to 15707)
  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 15707)
  AUTHORS   .
  TITLE     Direct Submission
  JOURNAL   Exported Dec 15, 2025 from SnapGene Server 8.0.1
            https://www.snapgene.com
COMMENT     SGRef: number: 1; type: "Journal Article"; journalName: "eLife."; 
            date: "2022-12-28"; volume: "11:e81856. doi"; pages: " 
            10.7554/eLife.81856"
COMMENT     Sequence Label: pHR-UCOE-EF1a-Zim3-dCas9-P2A-mCherry
FEATURES             Location/Qualifiers
     source          1..15707
                     /mol_type="other DNA"
                     /organism="synthetic DNA construct"
     promoter        complement(40..58)
                     /label=SP6 promoter
                     /note="promoter for bacteriophage SP6 RNA polymerase"
     primer_bind     complement(41..58)
                     /label=SP6
                     /note="SP6 promoter, forward primer"
     primer_bind     complement(348..367)
                     /label=pBRrevBam
                     /note="pBR322 vectors, tet region, downstream of BamHI, 
                     reverse primer"
     primer_bind     complement(581..600)
                     /label=pRS-marker
                     /note="pRS vectors, use to sequence yeast selectable 
                     marker"
     primer_bind     700..722
                     /label=pGEX 3'
                     /note="pGEX vectors, reverse primer"
     primer_bind     complement(760..778)
                     /label=pBRforEco
                     /note="pBR322 vectors, upsteam of EcoRI site, forward 
                     primer"
     promoter        846..950
                     /gene="bla"
                     /label=AmpR promoter
     CDS             join(951..1019,1020..1811)
                     /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(1169..1188)
                     /label=Amp-R
                     /note="Ampicillin resistance gene, reverse primer"
     rep_origin      1982..2570
                     /direction=RIGHT
                     /label=ori
                     /note="high-copy-number ColE1/pMB1/pBR322/pUC origin of 
                     replication"
     primer_bind     2471..2490
                     /label=pBR322ori-F
                     /note="pBR322 origin, forward primer"
     primer_bind     2724..2741
                     /label=L4440
                     /note="L4440 vector, forward primer"
     primer_bind     complement(2811..2831)
                     /label=pBABE 3'
                     /note="SV40 enhancer, reverse primer for pBABE vectors"
     promoter        2816..3145
                     /label=SV40 promoter
                     /note="SV40 enhancer and early promoter"
     rep_origin      2996..3131
                     /label=SV40 ori
                     /note="SV40 origin of replication"
     intron          4279..4344
                     /label=small t intron
                     /note="SV40 (simian virus 40) small t antigen intron"
     CDS             4474..4494
                     /codon_start=1
                     /product="nuclear localization signal of SV40 (simian virus
                     40) large T antigen"
                     /label=SV40 NLS
                     /translation="PKKKRKV"
     polyA_signal    4919..5053
                     /label=SV40 poly(A) signal
                     /note="SV40 polyadenylation signal"
     primer_bind     complement(4956..4975)
                     /label=SV40pA-R
                     /note="SV40 polyA, reverse primer"
     primer_bind     5010..5029
                     /label=EBV-rev
                     /note="SV40 polyA terminator, reverse primer"
     LTR             5221..5854
                     /label=3' LTR
                     /note="3' long terminal repeat (LTR) from HIV-1"
     misc_feature    5901..6026
                     /label=HIV-1 Psi
                     /note="packaging signal of human immunodeficiency virus 
                     type 1"
     misc_feature    6523..6756
                     /label=RRE
                     /note="The Rev response element (RRE) of HIV-1 allows for 
                     Rev-dependent mRNA export from the nucleus to the 
                     cytoplasm."
     CDS             6941..6985
                     /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    7270..7387
                     /label=cPPT/CTS
                     /note="central polypurine tract and central termination 
                     sequence of HIV-1"
     promoter        8119..9296
                     /label=EF-1-alpha promoter
                     /note="strong constitutive promoter for human elongation 
                     factor EF-1-alpha"
     intron          8350..9288
                     /label=EF-1-alpha intron A
                     /note="intron upstream of the start codon of human 
                     EF-1-alpha"
     primer_bind     9245..9265
                     /label=EF1a-F
                     /note="Human elongation factor-1a promoter, forward primer"
     protein_bind    complement(9653..9677)
                     /gene="mutant version of attB"
                     /label=attB2
                     /bound_moiety="BP Clonase(TM)"
                     /note="recombination site for the Gateway(R) BP reaction"
     CDS             9697..9717
                     /codon_start=1
                     /product="nuclear localization signal of SV40 (simian virus
                     40) large T antigen"
                     /label=SV40 NLS
                     /translation="PKKKRKV"
     regulatory      9766..9775
                     /label=Kozak sequence
                     /note="vertebrate consensus sequence for strong initiation 
                     of translation (Kozak, 1987)"
                     /regulatory_class="other"
     CDS             join(9772..9798,9799..9801,9802..12288,12289..12291,
                     12292..13875)
                     /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             9772..13875
                     /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             13879..13905
                     /codon_start=1
                     /product="HA (human influenza hemagglutinin) epitope tag"
                     /label=HA
                     /translation="YPYDVPDYA"
     CDS             13885..13911
                     /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             13924..13944
                     /codon_start=1
                     /product="nuclear localization signal of SV40 (simian virus
                     40) large T antigen"
                     /label=SV40 NLS
                     /translation="PKKKRKV"
     CDS             13951..13971
                     /codon_start=1
                     /product="nuclear localization signal of SV40 (simian virus
                     40) large T antigen"
                     /label=SV40 NLS
                     /translation="PKKKRKV"
     CDS             13981..14037
                     /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             14038..14745
                     /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(14180..14198)
                     /label=mCherry-R
                     /note="mCherry, reverse primer"
     primer_bind     14448..14467
                     /label=mCherry-F
                     /note="mCherry, forward primer"
     misc_feature    14791..15379
                     /label=WPRE
                     /note="woodchuck hepatitis virus posttranscriptional 
                     regulatory element"
     primer_bind     complement(14844..14864)
                     /label=WPRE-R
                     /note="WPRE, reverse primer"
     CDS             complement(15262..15273)
                     /codon_start=1
                     /product="Factor Xa recognition and cleavage site"
                     /label=Factor Xa site
                     /translation="IEGR"
     primer_bind     complement(15382..15398)
                     /label=KS primer
                     /note="common sequencing primer, one of multiple similar 
                     variants"
     primer_bind     complement(15383..15399)
                     /label=pBluescriptKS
                     /note="For pBluescript vector"
     LTR             join(15489..15707,1..15)
                     /label=3' LTR (Delta-U3)
                     /note="self-inactivating 3' long terminal repeat (LTR) from
                     HIV-1"
ORIGIN
        1 ggaaaatctc tagcagcatc tagaattaat tccgtgtatt ctatagtgtc acctaaatcg
       61 tatgtgtatg atacataagg ttatgtatta attgtagccg cgttctaacg acaatatgta
      121 caagcctaat tgtgtagcat ctggcttact gaagcagacc ctatcatctc tctcgtaaac
      181 tgccgtcaga gtcggtttgg ttggacgaac cttctgagtt tctggtaacg ccgtcccgca
      241 cccggaaatg gtcagcgaac caatcagcag ggtcatcgct agccagatcc tctacgccgg
      301 acgcatcgtg gccggcatca ccggcgccac aggtgcggtt gctggcgcct atatcgccga
      361 catcaccgat ggggaagatc gggctcgcca cttcgggctc atgagcgctt gtttcggcgt
      421 gggtatggtg gcaggccccg tggccggggg actgttgggc gccatctcct tgcatgcacc
      481 attccttgcg gcggcggtgc tcaacggcct caacctacta ctgggctgct tcctaatgca
      541 ggagtcgcat aagggagagc gtcgaatggt gcactctcag tacaatctgc tctgatgccg
      601 catagttaag ccagccccga cacccgccaa cacccgctga cgcgccctga cgggcttgtc
      661 tgctcccggc atccgcttac agacaagctg tgaccgtctc cgggagctgc atgtgtcaga
      721 ggttttcacc gtcatcaccg aaacgcgcga gacgaaaggg cctcgtgata cgcctatttt
      781 tataggttaa tgtcatgata ataatggttt cttagacgtc aggtggcact tttcggggaa
      841 atgtgcgcgg aacccctatt tgtttatttt tctaaataca ttcaaatatg tatccgctca
      901 tgagacaata accctgataa atgcttcaat aatattgaaa aaggaagagt atgagtattc
      961 aacatttccg tgtcgccctt attccctttt ttgcggcatt ttgccttcct gtttttgctc
     1021 acccagaaac gctggtgaaa gtaaaagatg ctgaagatca gttgggtgca cgagtgggtt
     1081 acatcgaact ggatctcaac agcggtaaga tccttgagag ttttcgcccc gaagaacgtt
     1141 ttccaatgat gagcactttt aaagttctgc tatgtggcgc ggtattatcc cgtattgacg
     1201 ccgggcaaga gcaactcggt cgccgcatac actattctca gaatgacttg gttgagtact
     1261 caccagtcac agaaaagcat cttacggatg gcatgacagt aagagaatta tgcagtgctg
     1321 ccataaccat gagtgataac actgcggcca acttacttct gacaacgatc ggaggaccga
     1381 aggagctaac cgcttttttg cacaacatgg gggatcatgt aactcgcctt gatcgttggg
     1441 aaccggagct gaatgaagcc ataccaaacg acgagcgtga caccacgatg cctgtagcaa
     1501 tggcaacaac gttgcgcaaa ctattaactg gcgaactact tactctagct tcccggcaac
     1561 aattaataga ctggatggag gcggataaag ttgcaggacc acttctgcgc tcggcccttc
     1621 cggctggctg gtttattgct gataaatctg gagccggtga gcgtgggtct cgcggtatca
     1681 ttgcagcact ggggccagat ggtaagccct cccgtatcgt agttatctac acgacgggga
     1741 gtcaggcaac tatggatgaa cgaaatagac agatcgctga gataggtgcc tcactgatta
     1801 agcattggta actgtcagac caagtttact catatatact ttagattgat ttaaaacttc
     1861 atttttaatt taaaaggatc taggtgaaga tcctttttga taatctcatg accaaaatcc
     1921 cttaacgtga gttttcgttc cactgagcgt cagaccccgt agaaaagatc aaaggatctt
     1981 cttgagatcc tttttttctg cgcgtaatct gctgcttgca aacaaaaaaa ccaccgctac
     2041 cagcggtggt ttgtttgccg gatcaagagc taccaactct ttttccgaag gtaactggct
     2101 tcagcagagc gcagatacca aatactgttc ttctagtgta gccgtagtta ggccaccact
     2161 tcaagaactc tgtagcaccg cctacatacc tcgctctgct aatcctgtta ccagtggctg
     2221 ctgccagtgg cgataagtcg tgtcttaccg ggttggactc aagacgatag ttaccggata
     2281 aggcgcagcg gtcgggctga acggggggtt cgtgcacaca gcccagcttg gagcgaacga
     2341 cctacaccga actgagatac ctacagcgtg agctatgaga aagcgccacg cttcccgaag
     2401 ggagaaaggc ggacaggtat ccggtaagcg gcagggtcgg aacaggagag cgcacgaggg
     2461 agcttccagg gggaaacgcc tggtatcttt atagtcctgt cgggtttcgc cacctctgac
     2521 ttgagcgtcg atttttgtga tgctcgtcag gggggcggag cctatggaaa aacgccagca
     2581 acgcggcctt tttacggttc ctggcctttt gctggccttt tgctcacatg ttctttcctg
     2641 cgttatcccc tgattctgtg gataaccgta ttaccgcctt tgagtgagct gataccgctc
     2701 gccgcagccg aacgaccgag cgcagcgagt cagtgagcga ggaagcggaa gagcgcccaa
     2761 tacgcaaacc gcctctcccc gcgcgttggc cgattcatta atgcagctgt ggaatgtgtg
     2821 tcagttaggg tgtggaaagt ccccaggctc cccagcaggc agaagtatgc aaagcatgca
     2881 tctcaattag tcagcaacca ggtgtggaaa gtccccaggc tccccagcag gcagaagtat
     2941 gcaaagcatg catctcaatt agtcagcaac catagtcccg cccctaactc cgcccatccc
     3001 gcccctaact ccgcccagtt ccgcccattc tccgccccat ggctgactaa ttttttttat
     3061 ttatgcagag gccgaggccg cctcggcctc tgagctattc cagaagtagt gaggaggctt
     3121 ttttggaggc ctaggctttt gcaaaaagct tggacacaag acaggcttgc gagatatgtt
     3181 tgagaatacc actttatccc gcgtcaggga gaggcagtgc gtaaaaagac gcggactcat
     3241 gtgaaatact ggtttttagt gcgccagatc tctataatct cgcgcaacct attttcccct
     3301 cgaacacttt ttaagccgta gataaacagg ctgggacact tcacatgagc gaaaaataca
     3361 tcgtcacctg ggacatgttg cagatccatg cacgtaaact cgcaagccga ctgatgcctt
     3421 ctgaacaatg gaaaggcatt attgccgtaa gccgtggcgg tctgtaccgg gtgcgttact
     3481 ggcgcgtgaa ctgggtattc gtcatgtcga taccgtttgt atttccagct acgatcacga
     3541 caaccagcgc gagcttaaag tgctgaaacg cgcagaaggc gatggcgaag gcttcatcgt
     3601 tattgatgac ctggtggata ccggtggtac tgcggttgcg attcgtgaaa tgtatccaaa
     3661 agcgcacttt gtcaccatct tcgcaaaacc ggctggtcgt ccgctggttg atgactatgt
     3721 tgttgatatc ccgcaagata cctggattga acagccgtgg gatatgggcg tcgtattcgt
     3781 cccgccaatc tccggtcgct aatcttttca acgcctggca ctgccgggcg ttgttctttt
     3841 taacttcagg cgggttacaa tagtttccag taagtattct ggaggctgca tccatgacac
     3901 aggcaaacct gagcgaaacc ctgttcaaac cccgctttaa acatcctgaa acctcgacgc
     3961 tagtccgccg ctttaatcac ggcgcacaac cgcctgtgca gtcggccctt gatggtaaaa
     4021 ccatccctca ctggtatcgc atgattaacc gtctgatgtg gatctggcgc ggcattgacc
     4081 cacgcgaaat cctcgacgtc caggcacgta ttgtgatgag cgatgccgaa cgtaccgacg
     4141 atgatttata cgatacggtg attggctacc gtggcggcaa ctggatttat gagtgggccc
     4201 cggatctttg tgaaggaacc ttacttctgt ggtgtgacat aattggacaa actacctaca
     4261 gagatttaaa gctctaaggt aaatataaaa tttttaagtg tataatgtgt taaactactg
     4321 attctaattg tttgtgtatt ttagattcca acctatggaa ctgatgaatg ggagcagtgg
     4381 tggaatgcct ttaatgagga aaacctgttt tgctcagaag aaatgccatc tagtgatgat
     4441 gaggctactg ctgactctca acattctact cctccaaaaa agaagagaaa ggtagaagac
     4501 cccaaggact ttccttcaga attgctaagt tttttgagtc atgctgtgtt tagtaataga
     4561 actcttgctt gctttgctat ttacaccaca aaggaaaaag ctgcactgct atacaagaaa
     4621 attatggaaa aatattctgt aacctttata agtaggcata acagttataa tcataacata
     4681 ctgttttttc ttactccaca caggcataga gtgtctgcta ttaataacta tgctcaaaaa
     4741 ttgtgtacct ttagcttttt aatttgtaaa ggggttaata aggaatattt gatgtatagt
     4801 gccttgacta gagatcataa tcagccatac cacatttgta gaggttttac ttgctttaaa
     4861 aaacctccca cacctccccc tgaacctgaa acataaaatg aatgcaattg ttgttgttaa
     4921 cttgtttatt gcagcttata atggttacaa ataaagcaat agcatcacaa atttcacaaa
     4981 taaagcattt ttttcactgc attctagttg tggtttgtcc aaactcatca atgtatctta
     5041 tcatgtctgg atcaactgga taactcaagc taaccaaaat catcccaaac ttcccacccc
     5101 ataccctatt accactgcca attacctgtg gtttcattta ctctaaacct gtgattcctc
     5161 tgaattattt tcattttaaa gaaattgtat ttgttaaata tgtactacaa acttagtagt
     5221 tggaagggct aattcactcc caaagaagac aagatatcct tgatctgtgg atctaccaca
     5281 cacaaggcta cttccctgat tagcagaact acacaccagg gccaggggtc agatatccac
     5341 tgacctttgg atggtgctac aagctagtac cagttgagcc agataaggta gaagaggcca
     5401 ataaaggaga gaacaccagc ttgttacacc ctgtgagcct gcatgggatg gatgacccgg
     5461 agagagaagt gttagagtgg aggtttgaca gccgcctagc atttcatcac gtggcccgag
     5521 agctgcatcc ggagtacttc aagaactgct gatatcgagc ttgctacaag ggactttccg
     5581 ctggggactt tccagggagg cgtggcctgg gcgggactgg ggagtggcga gccctcagat
     5641 cctgcatata agcagctgct ttttgcctgt actgggtctc tctggttaga ccagatctga
     5701 gcctgggagc tctctggcta actagggaac ccactgctta agcctcaata aagcttgcct
     5761 tgagtgcttc aagtagtgtg tgcccgtctg ttgtgtgact ctggtaacta gagatccctc
     5821 agaccctttt agtcagtgtg gaaaatctct agcagtggcg cccgaacagg gacttgaaag
     5881 cgaaagggaa accagaggag ctctctcgac gcaggactcg gcttgctgaa gcgcgcacgg
     5941 caagaggcga ggggcggcga ctggtgagta cgccaaaaat tttgactagc ggaggctaga
     6001 aggagagaga tgggtgcgag agcgtcagta ttaagcgggg gagaattaga tcgcgatggg
     6061 aaaaaattcg gttaaggcca gggggaaaga aaaaatataa attaaaacat atagtatggg
     6121 caagcaggga gctagaacga ttcgcagtta atcctggcct gttagaaaca tcagaaggct
     6181 gtagacaaat actgggacag ctacaaccat cccttcagac aggatcagaa gaacttagat
     6241 cattatataa tacagtagca accctctatt gtgtgcatca aaggatagag ataaaagaca
     6301 ccaaggaagc tttagacaag atagaggaag agcaaaacaa aagtaagacc accgcacagc
     6361 aagcggccgg ccgctgatct tcagacctgg aggaggagat atgagggaca attggagaag
     6421 tgaattatat aaatataaag tagtaaaaat tgaaccatta ggagtagcac ccaccaaggc
     6481 aaagagaaga gtggtgcaga gagaaaaaag agcagtggga ataggagctt tgttccttgg
     6541 gttcttggga gcagcaggaa gcactatggg cgcagcgtca atgacgctga cggtacaggc
     6601 cagacaatta ttgtctggta tagtgcagca gcagaacaat ttgctgaggg ctattgaggc
     6661 gcaacagcat ctgttgcaac tcacagtctg gggcatcaag cagctccagg caagaatcct
     6721 ggctgtggaa agatacctaa aggatcaaca gctcctgggg atttggggtt gctctggaaa
     6781 actcatttgc accactgctg tgccttggaa tgctagttgg agtaataaat ctctggaaca
     6841 gatttggaat cacacgacct ggatggagtg ggacagagaa attaacaatt acacaagctt
     6901 aatacactcc ttaattgaag aatcgcaaaa ccagcaagaa aagaatgaac aagaattatt
     6961 ggaattagat aaatgggcaa gtttgtggaa ttggtttaac ataacaaatt ggctgtggta
     7021 tataaaatta ttcataatga tagtaggagg cttggtaggt ttaagaatag tttttgctgt
     7081 actttctata gtgaatagag ttaggcaggg atattcacca ttatcgtttc agacccacct
     7141 cccaaccccg aggggacccg acaggcccga aggaatagaa gaagaaggtg gagagagaga
     7201 cagagacaga tccattcgat tagtgaacgg atctcgacgg tatcgccaaa tggcagtatt
     7261 catccacaat tttaaaagaa aaggggggat tggggggtac agtgcagggg aaagaatagt
     7321 agacataata gcaacagaca tacaaactaa agaattacaa aaacaaatta caaaaattca
     7381 aaattttcgg gtttattaca gggacagcag agatccagtt tggatcgata agcttgatat
     7441 cgaattcggg aggtggtccc tgcagttacg ccaatgataa cccccgccag aaaaatctta
     7501 gtagccttcc ctttttgttt tccgtgcccc aactcggcgg attgactcgg ccccttccgg
     7561 aaacacccga atcaacttct agtcaaatta ttgttcacgc cgcaatgacc cacccctggc
     7621 ccgcgtctgt ggaactgacc cctggtgtac aggagagttc gctgctgaaa gtggtcccaa
     7681 aggggtacta gtttttaagc tcccaactcc ccctccccca gcgtctggag gattccacac
     7741 cctcgcaccg caggggcgag gaagtgggcg gagtccggtt ttggcgccag ccgctgaggc
     7801 tgccaagcag aaaagccacc gctgaggaga ctccggtcac tgtcctcgcc ccgcctcccc
     7861 cttccctccc cttggggacc accgggcgcc acgccgcgaa cggtaagtgc cgcggtcgtc
     7921 ggcgcctccg ccctccccct agggccccaa ttcccagcgg gcgcggcgcg cggcccctcc
     7981 ccccgccggg cgcgcgcccg ctgccccgcc cttcgtggcc gcccggcgtg ggcggtgcca
     8041 cccctccccc cggcggcccc gcgcgcagct cccggctccc tcccccttcg gatgtggctt
     8101 gagctgtagg cgcggagggg ctccggtgcc cgtcagtggg cagagcgcac atcgcccaca
     8161 gtccccgaga agttgggggg aggggtcggc aattgaaccg gtgcctagag aaggtggcgc
     8221 ggggtaaact gggaaagtga tgtcgtgtac tggctccgcc tttttcccga gggtggggga
     8281 gaaccgtata taagtgcagt agtcgccgtg aacgttcttt ttcgcaacgg gtttgccgcc
     8341 agaacacagg taagtgccgt gtgtggttcc cgcgggcctg gcctctttac gggttatggc
     8401 ccttgcgtgc cttgaattac ttccacctgg ctgcagtacg tgattcttga tcccgagctt
     8461 cgggttggaa gtgggtggga gagttcgagg ccttgcgctt aaggagcccc ttcgcctcgt
     8521 gcttgagttg aggcctggcc tgggcgctgg ggccgccgcg tgcgaatctg gtggcacctt
     8581 cgcgcctgtc tcgctgcttt cgataagtct ctagccattt aaaatttttg atgacctgct
     8641 gcgacgcttt ttttctggca agatagtctt gtaaatgcgg gccaagatct gcacactggt
     8701 atttcggttt ttggggccgc gggcggcgac ggggcccgtg cgtcccagcg cacatgttcg
     8761 gcgaggcggg gcctgcgagc gcggccaccg agaatcggac gggggtagtc tcaagctggc
     8821 cggcctgctc tggtgcctgg cctcgcgccg ccgtgtatcg ccccgccctg ggcggcaagg
     8881 ctggcccggt cggcaccagt tgcgtgagcg gaaagatggc cgcttcccgg ccctgctgca
     8941 gggagctcaa aatggaggac gcggcgctcg ggagagcggg cgggtgagtc acccacacaa
     9001 aggaaaaggg cctttccgtc ctcagccgtc gcttcatgtg actccacgga gtaccgggcg
     9061 ccgtccaggc acctcgatta gttctcgagc ttttggagta cgtcgtcttt aggttggggg
     9121 gaggggtttt atgcgatgga gtttccccac actgagtggg tggagactga agttaggcca
     9181 gcttggcact tgatgtaatt ctccttggaa tttgcccttt ttgagtttgg atcttggttc
     9241 attctcaagc ctcagacagt ggttcaaagt ttttttcttc catttcaggt gtcgtggaat
     9301 tctcacgcgt ctgcaggata tcaagcttgc ggtaccgcgg gcccggccac catgaacaat
     9361 tcccagggaa gagtgacctt cgaggatgtc actgtgaact tcacccaggg ggagtggcag
     9421 cggctgaatc ccgaacagag aaacttgtac agggatgtga tgctggagaa ttacagcaac
     9481 cttgtctctg tgggacaagg ggaaaccacc aaacccgatg tgatcttgag gttggaacaa
     9541 ggaaaggagc catggttgga ggaagaggaa gtgctgggaa gtggccgtgc agaaaaaaat
     9601 ggggacattg gagggcagat ttggaagcca aaggatgtga aagagagtct ctacccaact
     9661 ttcttgtaca aagtggttgg tggatctgga ggttctccca agaagaagcg caaggtggga
     9721 cgcgtctgca ggatatcaag cttgcggtac cgcgggcccg ggatcgccac catggacaag
     9781 aagtacagca tcggcctggc catcggcacc aactctgtgg gctgggccgt gatcaccgac
     9841 gagtacaagg tgcccagcaa gaaattcaag gtgctgggca acaccgaccg gcacagcatc
     9901 aagaagaacc tgatcggcgc cctgctgttc gacagcggag aaacagccga ggccacccgg
     9961 ctgaagagaa ccgccagaag aagatacacc agacggaaga accggatctg ctatctgcaa
    10021 gagatcttca gcaacgagat ggccaaggtg gacgacagct tcttccacag actggaagag
    10081 tccttcctgg tggaagagga taagaagcac gagcggcacc ccatcttcgg caacatcgtg
    10141 gacgaggtgg cctaccacga gaagtacccc accatctacc acctgagaaa gaaactggtg
    10201 gacagcaccg acaaggccga cctgcggctg atctatctgg ccctggccca catgatcaag
    10261 ttccggggcc acttcctgat cgagggcgac ctgaaccccg acaacagcga cgtggacaag
    10321 ctgttcatcc agctggtgca gacctacaac cagctgttcg aggaaaaccc catcaacgcc
    10381 agcggcgtgg acgccaaggc catcctgtct gccagactga gcaagagcag acggctggaa
    10441 aatctgatcg cccagctgcc cggcgagaag aagaatggcc tgttcggcaa cctgattgcc
    10501 ctgagcctgg gcctgacccc caacttcaag agcaacttcg acctggccga ggatgccaaa
    10561 ctgcagctga gcaaggacac ctacgacgac gacctggaca acctgctggc ccagatcggc
    10621 gaccagtacg ccgacctgtt tctggccgcc aagaacctgt ccgacgccat cctgctgagc
    10681 gacatcctga gagtgaacac cgagatcacc aaggcccccc tgagcgcctc tatgatcaag
    10741 agatacgacg agcaccacca ggacctgacc ctgctgaaag ctctcgtgcg gcagcagctg
    10801 cctgagaagt acaaagagat tttcttcgac cagagcaaga acggctacgc cggctacatc
    10861 gatggcggag ccagccagga agagttctac aagttcatca agcccatcct ggaaaagatg
    10921 gacggcaccg aggaactgct cgtgaagctg aacagagagg acctgctgcg gaagcagcgg
    10981 accttcgaca acggcagcat cccccaccag atccacctgg gagagctgca cgccattctg
    11041 cggcggcagg aagattttta cccattcctg aaggacaacc gggaaaagat cgagaagatc
    11101 ctgaccttcc gcatccccta ctacgtgggc cctctggcca ggggaaacag cagattcgcc
    11161 tggatgacca gaaagagcga ggaaaccatc accccctgga acttcgagga agtggtggac
    11221 aagggcgcca gcgcccagag cttcatcgag cggatgacca acttcgataa gaacctgccc
    11281 aacgagaagg tgctgcccaa gcacagcctg ctgtacgagt acttcaccgt gtacaacgag
    11341 ctgaccaaag tgaaatacgt gaccgaggga atgagaaagc ccgccttcct gagcggcgag
    11401 cagaaaaaag ccatcgtgga cctgctgttc aagaccaacc ggaaagtgac cgtgaagcag
    11461 ctgaaagagg actacttcaa gaaaatcgag tgcttcgact ccgtggaaat ctccggcgtg
    11521 gaagatcggt tcaacgcctc cctgggcaca taccacgatc tgctgaaaat tatcaaggac
    11581 aaggacttcc tggacaatga ggaaaacgag gacattctgg aagatatcgt gctgaccctg
    11641 acactgtttg aggacagaga gatgatcgag gaacggctga aaacctatgc ccacctgttc
    11701 gacgacaaag tgatgaagca gctgaagcgg cggagataca ccggctgggg caggctgagc
    11761 cggaagctga tcaacggcat ccgggacaag cagtccggca agacaatcct ggatttcctg
    11821 aagtccgacg gcttcgccaa cagaaacttc atgcagctga tccacgacga cagcctgacc
    11881 tttaaagagg acatccagaa agcccaggtg tccggccagg gcgatagcct gcacgagcac
    11941 attgccaatc tggccggcag ccccgccatt aagaagggca tcctgcagac agtgaaggtg
    12001 gtggacgagc tcgtgaaagt gatgggccgg cacaagcccg agaacatcgt gatcgaaatg
    12061 gccagagaga accagaccac ccagaaggga cagaagaaca gccgcgagag aatgaagcgg
    12121 atcgaagagg gcatcaaaga gctgggcagc cagatcctga aagaacaccc cgtggaaaac
    12181 acccagctgc agaacgagaa gctgtacctg tactacctgc agaatgggcg ggatatgtac
    12241 gtggaccagg aactggacat caaccggctg tccgactacg atgtggacgc tatcgtgcct
    12301 cagagctttc tgaaggacga ctccatcgat aacaaagtgc tgactcggag cgacaagaac
    12361 cggggcaaga gcgacaacgt gccctccgaa gaggtcgtga agaagatgaa gaactactgg
    12421 cgccagctgc tgaatgccaa gctgattacc cagaggaagt tcgacaatct gaccaaggcc
    12481 gagagaggcg gcctgagcga actggataag gccggcttca tcaagagaca gctggtggaa
    12541 acccggcaga tcacaaagca cgtggcacag atcctggact cccggatgaa cactaagtac
    12601 gacgagaacg acaaactgat ccgggaagtg aaagtgatca ccctgaagtc caagctggtg
    12661 tccgatttcc ggaaggattt ccagttttac aaagtgcgcg agatcaacaa ctaccaccac
    12721 gcccacgacg cctacctgaa cgccgtcgtg ggaaccgccc tgatcaaaaa gtaccctaag
    12781 ctggaaagcg agttcgtgta cggcgactac aaggtgtacg acgtgcggaa gatgatcgcc
    12841 aagagcgagc aggaaatcgg caaggctacc gccaagtact tcttctacag caacatcatg
    12901 aactttttca agaccgagat taccctggcc aacggcgaga tccggaagcg gcctctgatc
    12961 gagacaaacg gcgaaacagg cgagatcgtg tgggataagg gccgggactt tgccaccgtg
    13021 cggaaagtgc tgtctatgcc ccaagtgaat atcgtgaaaa agaccgaggt gcagacaggc
    13081 ggcttcagca aagagtctat cctgcccaag aggaacagcg acaagctgat cgccagaaag
    13141 aaggactggg accctaagaa gtacggcggc ttcgacagcc ccaccgtggc ctattctgtg
    13201 ctggtggtgg ccaaagtgga aaagggcaag tccaagaaac tgaagagtgt gaaagagctg
    13261 ctggggatca ccatcatgga aagaagcagc ttcgagaaga atcccatcga ctttctggaa
    13321 gccaagggct acaaagaagt gaaaaaggac ctgatcatca agctgcctaa gtactccctg
    13381 ttcgagctgg aaaacggccg gaagagaatg ctggcctctg ccggcgaact gcagaaggga
    13441 aacgaactgg ccctgccctc caaatatgtg aacttcctgt acctggccag ccactatgag
    13501 aagctgaagg gctcccccga ggataatgag cagaaacagc tgtttgtgga acagcacaaa
    13561 cactacctgg acgagatcat cgagcagatc agcgagttct ccaagagagt gatcctggcc
    13621 gacgctaatc tggacaaggt gctgagcgcc tacaacaagc acagagacaa gcctatcaga
    13681 gagcaggccg agaatatcat ccacctgttt accctgacca atctgggagc ccctgccgcc
    13741 ttcaagtact ttgacaccac catcgaccgg aagaggtaca ccagcaccaa agaggtgctg
    13801 gacgccaccc tgatccacca gagcatcacc ggcctgtacg agacacggat cgacctgtct
    13861 cagctgggag gcgacgccta tccctatgac gtgcccgatt atgccagcct gggcagcggc
    13921 tcccccaaga aaaaacgcaa ggtggaagat cctaagaaaa agcggaaagt gagcggccgc
    13981 gctactaact tcagcctgct gaagcaggct ggagacgtgg aggagaaccc tggacctgtg
    14041 agcaagggcg aggaggataa catggccatc atcaaggagt tcatgcgctt caaggtgcac
    14101 atggagggct ccgtgaacgg ccacgagttc gagatcgagg gcgagggcga gggccgcccc
    14161 tacgagggca cccagaccgc caagctgaag gtgaccaagg gcggccccct gcccttcgcc
    14221 tgggacatcc tgtcccctca gttcatgtac ggctccaagg cctacgtgaa gcaccccgcc
    14281 gacatccccg actacttgaa gctgtccttc cccgagggct tcaagtggga gcgcgtgatg
    14341 aacttcgagg acggcggcgt ggtgaccgtg acccaggact cctccctgca ggacggcgag
    14401 ttcatctaca aggtgaagct gcgcggcacc aacttcccct ccgacggccc cgtaatgcag
    14461 aagaagacca tgggctggga ggcctcctcc gagcggatgt accccgagga cggcgccctg
    14521 aagggcgaga tcaagcagag gctgaagctg aaggacggcg gccactacga cgctgaggtc
    14581 aagaccacct acaaggccaa gaagcccgtg cagctgcccg gcgcctacaa cgtcaacatc
    14641 aagttggaca tcacctccca caacgaggac tacaccatcg tggaacagta cgaacgcgcc
    14701 gagggccgcc actccaccgg cggcatggac gagctgtaca agtaagcgat cgccctgcag
    14761 gcatgcaagc ttgatatcaa gcttatcgat aatcaacctc tggattacaa aatttgtgaa
    14821 agattgactg gtattcttaa ctatgttgct ccttttacgc tatgtggata cgctgcttta
    14881 atgcctttgt atcatgctat tgcttcccgt atggctttca ttttctcctc cttgtataaa
    14941 tcctggttgc tgtctcttta tgaggagttg tggcccgttg tcaggcaacg tggcgtggtg
    15001 tgcactgtgt ttgctgacgc aacccccact ggttggggca ttgccaccac ctgtcagctc
    15061 ctttccggga ctttcgcttt ccccctccct attgccacgg cggaactcat cgccgcctgc
    15121 cttgcccgct gctggacagg ggctcggctg ttgggcactg acaattccgt ggtgttgtcg
    15181 gggaaatcat cgtcctttcc ttggctgctc gcctgtgttg ccacctggat tctgcgcggg
    15241 acgtccttct gctacgtccc ttcggccctc aatccagcgg accttccttc ccgcggcctg
    15301 ctgccggctc tgcggcctct tccgcgtctt cgccttcgcc ctcagacgag tcggatctcc
    15361 ctttgggccg cctccccgca tcgataccgt cgacctcgag ggaattaatt cgagctcggt
    15421 acctttaaga ccaatgactt acaaggcagc tgtagatctt agccactttt taaaagaaaa
    15481 ggggggactg gaagggctaa ttcactccca acgaagacaa gatctgcttt ttgcttgtac
    15541 tgggtctctc tggttagacc agatctgagc ctgggagctc tctggctaac tagggaaccc
    15601 actgcttaag cctcaataaa gcttgccttg agtgcttcaa gtagtgtgtg cccgtctgtt
    15661 gtgtgactct ggtaactaga gatccctcag acccttttag tcagtgt
//
