LOCUS       sequence-178373-       15807 bp    DNA     circular SYN 15-DEC-2025
DEFINITION  for CRISPRi, lentiviral expression of dCas9-p300 2A BlastR..
ACCESSION   .
VERSION     .
KEYWORDS    .
SOURCE      synthetic DNA construct
  ORGANISM  synthetic DNA construct
REFERENCE   1  (bases 1 to 15807)
  AUTHORS   Najm FJ, Strand C, Donovan KF, Hegde M, Sanson KR, Vaimberg EW, 
            Sullender ME, Hartenian E, Kalani Z, Fusi N, Listgarten J, Younger 
            ST, Bernstein BE, Root DE, Doench JG
  TITLE     Orthologous CRISPR-Cas9 enzymes for combinatorial genetic screens.
  JOURNAL   Nat Biotechnol. 2018 Feb;36(2):179-189. doi: 10.1038/nbt.4048. Epub 
            2017 Dec 18.
  PUBMED    29251726
REFERENCE   2  (bases 1 to 15807)
  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"; doi: "10.1038/nbt.4048"; 
            journalName: "Nat Biotechnol"; date: "2018-02"; volume: "36"; issue:
            "2"; pages: "179-189"
COMMENT     Sequence Label: pXPR_123
FEATURES             Location/Qualifiers
     source          1..15807
                     /mol_type="other DNA"
                     /organism="synthetic DNA construct"
     primer_bind     complement(47..66)
                     /label=pRS-marker
                     /note="pRS vectors, use to sequence yeast selectable 
                     marker"
     enhancer        238..617
                     /label=CMV enhancer
                     /note="human cytomegalovirus immediate early enhancer"
     promoter        619..817
                     /label=CMV promoter
                     /note="human cytomegalovirus (CMV) immediate early 
                     promoter"
     primer_bind     772..792
                     /label=CMV-F
                     /note="Human CMV immediate early promoter, forward primer"
     LTR             835..1015
                     /label=5' LTR (truncated)
                     /note="truncated 5' long terminal repeat (LTR) from HIV-1"
     misc_feature    1062..1187
                     /label=HIV-1 Psi
                     /note="packaging signal of human immunodeficiency virus 
                     type 1"
     misc_feature    1680..1913
                     /label=RRE
                     /note="The Rev response element (RRE) of HIV-1 allows for 
                     Rev-dependent mRNA export from the nucleus to the 
                     cytoplasm."
     CDS             2098..2142
                     /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    2440..2557
                     /label=cPPT/CTS
                     /note="central polypurine tract and central termination 
                     sequence of HIV-1"
     promoter        2687..3865
                     /label=EF-1-alpha promoter
                     /note="strong constitutive promoter for human elongation 
                     factor EF-1-alpha"
     intron          2918..3856
                     /label=EF-1-alpha intron A
                     /note="intron upstream of the start codon of human 
                     EF-1-alpha"
     primer_bind     3813..3833
                     /label=EF1a-F
                     /note="Human elongation factor-1a promoter, forward primer"
     CDS             3881..3928
                     /codon_start=1
                     /product="bipartite nuclear localization signal from 
                     nucleoplasmin"
                     /label=nucleoplasmin NLS
                     /translation="KRPAATKKAGQAKKKK"
     CDS             join(3929..3952,3953..3955,3956..6511,6512..6514,
                     6515..8029)
                     /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 N863A mutation in the HNH 
                     catalytic domain"
                     /translation="DKKYSIGLAIGTNSVGWAVITDEYKVPSKKFKVLGNTDRHSIKKN
                     LIGALLFDSGETAEATRLKRTARRRYTRRKNRICYLQEIFSNEMAKVDDSFFHRLEESF
                     LVEEDKKHERHPIFGNIVDEVAYHEKYPTIYHLRKKLVDSTDKADLRLIYLALAHMIKF
                     RGHFLIEGDLNPDNSDVDKLFIQLVQTYNQLFEENPINASGVDAKAILSARLSKSRRLE
                     NLIAQLPGEKKNGLFGNLIALSLGLTPNFKSNFDLAEDAKLQLSKDTYDDDLDNLLAQI
                     GDQYADLFLAAKNLSDAILLSDILRVNTEITKAPLSASMIKRYDEHHQDLTLLKALVRQ
                     QLPEKYKEIFFDQSKNGYAGYIDGGASQEEFYKFIKPILEKMDGTEELLVKLNREDLLR
                     KQRTFDNGSIPHQIHLGELHAILRRQEDFYPFLKDNREKIEKILTFRIPYYVGPLARGN
                     SRFAWMTRKSEETITPWNFEEVVDKGASAQSFIERMTNFDKNLPNEKVLPKHSLLYEYF
                     TVYNELTKVKYVTEGMRKPAFLSGEQKKAIVDLLFKTNRKVTVKQLKEDYFKKIECFDS
                     VEISGVEDRFNASLGTYHDLLKIIKDKDFLDNEENEDILEDIVLTLTLFEDREMIEERL
                     KTYAHLFDDKVMKQLKRRRYTGWGRLSRKLINGIRDKQSGKTILDFLKSDGFANRNFMQ
                     LIHDDSLTFKEDIQKAQVSGQGDSLHEHIANLAGSPAIKKGILQTVKVVDELVKVMGRH
                     KPENIVIEMARENQTTQKGQKNSRERMKRIEEGIKELGSQILKEHPVENTQLQNEKLYL
                     YYLQNGRDMYVDQELDINRLSDYDVDHIVPQSFLKDDSIDNKVLTRSDKARGKSDNVPS
                     EEVVKKMKNYWRQLLNAKLITQRKFDNLTKAERGGLSELDKAGFIKRQLVETRQITKHV
                     AQILDSRMNTKYDENDKLIREVKVITLKSKLVSDFRKDFQFYKVREINNYHHAHDAYLN
                     AVVGTALIKKYPKLESEFVYGDYKVYDVRKMIAKSEQEIGKATAKYFFYSNIMNFFKTE
                     ITLANGEIRKRPLIETNGETGEIVWDKGRDFATVRKVLSMPQVNIVKKTEVQTGGFSKE
                     SILPKRNSDKLIARKKDWDPKKYGGFDSPTVAYSVLVVAKVEKGKSKKLKSVKELLGIT
                     IMERSSFEKNPIDFLEAKGYKEVKKDLIIKLPKYSLFELENGRKRMLASAGELQKGNEL
                     ALPSKYVNFLYLASHYEKLKGSPEDNEQKQLFVEQHKHYLDEIIEQISEFSKRVILADA
                     NLDKVLSAYNKHRDKPIREQAENIIHLFTLTNLGAPAAFKYFDTTIDRKRYTSTKEVLD
                     ATLIHQSITGLYETRIDLSQLGGD"
     CDS             8084..8104
                     /codon_start=1
                     /product="nuclear localization signal of SV40 (simian virus
                     40) large T antigen"
                     /label=SV40 NLS
                     /translation="PKKKRKV"
     CDS             8120..9970
                     /codon_start=1
                     /gene="EP300"
                     /product="catalytic histone acetyltransferase core of the 
                     human E1A-associated protein p300 (Delvecchio et al., 
                     2013)"
                     /label=p300 core
                     /translation="IFKPEELRQALMPTLEALYRQDPESLPFRQPVDPQLLGIPDYFDI
                     VKSPMDLSTIKRKLDTGQYQEPWQYVDDIWLMFNNAWLYNRKTSRVYKYCSKLSEVFEQ
                     EIDPVMQSLGYCCGRKLEFSPQTLCCYGKQLCTIPRDATYYSYQNRYHFCEKCFNEIQG
                     ESVSLGDDPSQPQTTINKEQFSKRKNDTLDPELFVECTECGRKMHQICVLHHEIIWPAG
                     FVCDGCLKKSARTRKENKFSAKRLPSTRLGTFLENRVNDFLRRQNHPESGEVTVRVVHA
                     SDKTVEVKPGMKARFVDSGEMAESFPYRTKALFAFEEIDGVDLCFFGMHVQEYGSDCPP
                     PNQRRVYISYLDSVHFFRPKCLRTAVYHEILIGYLEYVKKLGYTTGHIWACPPSEGDDY
                     IFHCHPPDQKIPKPKRLQEWYKKMLDKAVSERIVHDYKDIFKQATEDRLTSAKELPYFE
                     GDFWPNVLEESIKELEQEEEERKREENTSNESTDVTKGDSKNAKKKNNKKTSKNKSSLS
                     RGNKKKPGMPNVSNDLSQKLYATMEKHKEVFFVIRLIAGPAANSLPPIVDPDPLIPCDL
                     MDGRDAFLTLARDKHLEFSSLRRAQWSTMCMLVELHTQSQD"
     regulatory      9734..9743
                     /label=Kozak sequence
                     /note="vertebrate consensus sequence for strong initiation 
                     of translation (Kozak, 1987)"
                     /regulatory_class="other"
     CDS             9971..9997
                     /codon_start=1
                     /product="HA (human influenza hemagglutinin) epitope tag"
                     /label=HA
                     /translation="YPYDVPDYA"
     CDS             10022..10075
                     /codon_start=1
                     /product="2A peptide from Thosea asigna virus capsid 
                     protein"
                     /label=T2A
                     /note="Eukaryotic ribosomes fail to insert a peptide bond 
                     between the Gly and Pro residues, yielding separate 
                     polypeptides."
                     /translation="EGRGSLLTCGDVEENPGP"
     CDS             10076..10474
                     /codon_start=1
                     /gene="Aspergillus terreus BSD"
                     /product="blasticidin S deaminase"
                     /label=BSD
                     /note="confers resistance to blasticidin"
                     /translation="MAKPLSQEESTLIERATATINSIPISEDYSVASAALSSDGRIFTG
                     VNVYHFTGGPCAELVVLGTAAAAAAGNLTCIVAIGNENRGILSPCGRCRQVLLDLHPGI
                     KAIVKDSDGQPTAVGIRELLPSGYVWEG"
     misc_feature    10499..11087
                     /label=WPRE
                     /note="woodchuck hepatitis virus posttranscriptional 
                     regulatory element"
     primer_bind     complement(10552..10572)
                     /label=WPRE-R
                     /note="WPRE, reverse primer"
     CDS             complement(10970..10981)
                     /codon_start=1
                     /product="Factor Xa recognition and cleavage site"
                     /label=Factor Xa site
                     /translation="IEGR"
     primer_bind     complement(11090..11106)
                     /label=KS primer
                     /note="common sequencing primer, one of multiple similar 
                     variants"
     primer_bind     complement(11091..11107)
                     /label=pBluescriptKS
                     /note="For pBluescript vector"
     LTR             11612..11792
                     /label=5' LTR (truncated)
                     /note="truncated 5' long terminal repeat (LTR) from HIV-1"
     primer_bind     complement(11818..11835)
                     /label=BGH-rev
                     /note="Bovine growth hormone terminator, reverse primer. 
                     Also called BGH reverse"
     polyA_signal    11824..12048
                     /label=bGH poly(A) signal
                     /note="bovine growth hormone polyadenylation signal"
     rep_origin      12094..12522
                     /direction=RIGHT
                     /label=f1 ori
                     /note="f1 bacteriophage origin of replication; arrow 
                     indicates direction of (+) strand synthesis"
     primer_bind     complement(12181..12200)
                     /label=F1ori-R
                     /note="F1 origin, reverse primer"
     primer_bind     12391..12412
                     /label=F1ori-F
                     /note="F1 origin, forward primer"
     primer_bind     complement(12531..12551)
                     /label=pBABE 3'
                     /note="SV40 enhancer, reverse primer for pBABE vectors"
     promoter        12536..12865
                     /label=SV40 promoter
                     /note="SV40 enhancer and early promoter"
     rep_origin      12716..12851
                     /label=SV40 ori
                     /note="SV40 origin of replication"
     primer_bind     12778..12797
                     /label=SV40pro-F
                     /note="SV40 promoter/origin, forward primer"
     promoter        12913..12960
                     /label=EM7 promoter
                     /note="synthetic bacterial promoter "
     CDS             12979..13353
                     /codon_start=1
                     /gene="Sh ble from Streptoalloteichus hindustanus"
                     /product="antibiotic-binding protein"
                     /label=BleoR
                     /note="confers resistance to bleomycin, phleomycin, and 
                     Zeocin(TM)"
                     /translation="MAKLTSAVPVLTARDVAGAVEFWTDRLGFSRDFVEDDFAGVVRDD
                     VTLFISAVQDQVVPDNTLAWVWVRGLDELYAEWSEVVSTNFRDASGPAMTEIGEQPWGR
                     EFALRDPAGNCVHFVAEEQD"
     polyA_signal    13483..13604
                     /label=SV40 poly(A) signal
                     /note="SV40 polyadenylation signal"
     primer_bind     complement(13520..13539)
                     /label=SV40pA-R
                     /note="SV40 polyA, reverse primer"
     primer_bind     13574..13593
                     /label=EBV-rev
                     /note="SV40 polyA terminator, reverse primer"
     primer_bind     complement(13653..13669)
                     /label=M13 rev
                     /note="common sequencing primer, one of multiple similar 
                     variants"
     primer_bind     complement(13653..13669)
                     /label=M13 Reverse
                     /note="In lacZ gene. Also called M13-rev"
     primer_bind     complement(13666..13688)
                     /label=M13/pUC Reverse
                     /note="In lacZ gene"
     protein_bind    13677..13693
                     /label=lac operator
                     /bound_moiety="lac repressor encoded by lacI"
                     /note="The lac repressor binds to the lac operator to 
                     inhibit transcription in E. coli. This inhibition can be 
                     relieved by adding lactose or 
                     isopropyl-beta-D-thiogalactopyranoside (IPTG)."
     promoter        complement(join(13701..13707,13708..13725,13726..13731))
                     /label=lac promoter
                     /note="promoter for the E. coli lac operon"
     protein_bind    13746..13767
                     /label=CAP binding site
                     /bound_moiety="E. coli catabolite activator protein"
                     /note="CAP binding activates transcription in the presence 
                     of cAMP."
     primer_bind     complement(13884..13901)
                     /label=L4440
                     /note="L4440 vector, forward primer"
     rep_origin      complement(14055..14643)
                     /direction=LEFT
                     /label=ori
                     /note="high-copy-number ColE1/pMB1/pBR322/pUC origin of 
                     replication"
     primer_bind     complement(14135..14154)
                     /label=pBR322ori-F
                     /note="pBR322 origin, forward primer"
     CDS             complement(join(14814..15605,15606..15674))
                     /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     15437..15456
                     /label=Amp-R
                     /note="Ampicillin resistance gene, reverse primer"
     promoter        complement(15675..15779)
                     /gene="bla"
                     /label=AmpR promoter
ORIGIN
        1 gtcgacggat cgggagatct cccgatcccc tatggtgcac tctcagtaca atctgctctg
       61 atgccgcata gttaagccag tatctgctcc ctgcttgtgt gttggaggtc gctgagtagt
      121 gcgcgagcaa aatttaagct acaacaaggc aaggcttgac cgacaattgc atgaagaatc
      181 tgcttagggt taggcgtttt gcgctgcttc gcgatgtacg ggccagatat acgcgttgac
      241 attgattatt gactagttat taatagtaat caattacggg gtcattagtt catagcccat
      301 atatggagtt ccgcgttaca taacttacgg taaatggccc gcctggctga ccgcccaacg
      361 acccccgccc attgacgtca ataatgacgt atgttcccat agtaacgcca atagggactt
      421 tccattgacg tcaatgggtg gagtatttac ggtaaactgc ccacttggca gtacatcaag
      481 tgtatcatat gccaagtacg ccccctattg acgtcaatga cggtaaatgg cccgcctggc
      541 attatgccca gtacatgacc ttatgggact ttcctacttg gcagtacatc tacgtattag
      601 tcatcgctat taccatggtg atgcggtttt ggcagtacat caatgggcgt ggatagcggt
      661 ttgactcacg gggatttcca agtctccacc ccattgacgt caatgggagt ttgttttggc
      721 accaaaatca acgggacttt ccaaaatgtc gtaacaactc cgccccattg acgcaaatgg
      781 gcggtaggcg tgtacggtgg gaggtctata taagcagcgc gttttgcctg tactgggtct
      841 ctctggttag accagatctg agcctgggag ctctctggct aactagggaa cccactgctt
      901 aagcctcaat aaagcttgcc ttgagtgctt caagtagtgt gtgcccgtct gttgtgtgac
      961 tctggtaact agagatccct cagacccttt tagtcagtgt ggaaaatctc tagcagtggc
     1021 gcccgaacag ggacttgaaa gcgaaaggga aaccagagga gctctctcga cgcaggactc
     1081 ggcttgctga agcgcgcacg gcaagaggcg aggggcggcg actggtgagt acgccaaaaa
     1141 ttttgactag cggaggctag aaggagagag atgggtgcga gagcgtcagt attaagcggg
     1201 ggagaattag atcgcgatgg gaaaaaattc ggttaaggcc agggggaaag aaaaaatata
     1261 aattaaaaca tatagtatgg gcaagcaggg agctagaacg attcgcagtt aatcctggcc
     1321 tgttagaaac atcagaaggc tgtagacaaa tactgggaca gctacaacca tcccttcaga
     1381 caggatcaga agaacttaga tcattatata atacagtagc aaccctctat tgtgtgcatc
     1441 aaaggataga gataaaagac accaaggaag ctttagacaa gatagaggaa gagcaaaaca
     1501 aaagtaagac caccgcacag caagcggccg ctgatcttca gacctggagg aggagatatg
     1561 agggacaatt ggagaagtga attatataaa tataaagtag taaaaattga accattagga
     1621 gtagcaccca ccaaggcaaa gagaagagtg gtgcagagag aaaaaagagc agtgggaata
     1681 ggagctttgt tccttgggtt cttgggagca gcaggaagca ctatgggcgc agcgtcaatg
     1741 acgctgacgg tacaggccag acaattattg tctggtatag tgcagcagca gaacaatttg
     1801 ctgagggcta ttgaggcgca acagcatctg ttgcaactca cagtctgggg catcaagcag
     1861 ctccaggcaa gaatcctggc tgtggaaaga tacctaaagg atcaacagct cctggggatt
     1921 tggggttgct ctggaaaact catttgcacc actgctgtgc cttggaatgc tagttggagt
     1981 aataaatctc tggaacagat ttggaatcac acgacctgga tggagtggga cagagaaatt
     2041 aacaattaca caagcttaat acactcctta attgaagaat cgcaaaacca gcaagaaaag
     2101 aatgaacaag aattattgga attagataaa tgggcaagtt tgtggaattg gtttaacata
     2161 acaaattggc tgtggtatat aaaattattc ataatgatag taggaggctt ggtaggttta
     2221 agaatagttt ttgctgtact ttctatagtg aatagagtta ggcagggata ttcaccatta
     2281 tcgtttcaga cccacctccc aaccccgagg ggacccgaca ggcccgaagg aatagaagaa
     2341 gaaggtggag agagagacag agacagatcc attcgattag tgaacggatc ggcactgcgt
     2401 gcgccaattc tgcagacaaa tggcagtatt catccacaat tttaaaagaa aaggggggat
     2461 tggggggtac agtgcagggg aaagaatagt agacataata gcaacagaca tacaaactaa
     2521 agaattacaa aaacaaatta caaaaattca aaattttcgg gtttattaca gggacagcag
     2581 agatccagtt tggttaatta gctagctgca aagatggata aagttttaaa cagagaggaa
     2641 tctttgcagc taatggacct tctaggtctt gaaaggagtg ggaattggct ccggtgcccg
     2701 tcagtgggca gagcgcacat cgcccacagt ccccgagaag ttggggggag gggtcggcaa
     2761 ttgaaccggt gcctagagaa ggtggcgcgg ggtaaactgg gaaagtgatg tcgtgtactg
     2821 gctccgcctt tttcccgagg gtgggggaga accgtatata agtgcagtag tcgccgtgaa
     2881 cgttcttttt cgcaacgggt ttgccgccag aacacaggta agtgccgtgt gtggttcccg
     2941 cgggcctggc ctctttacgg gttatggccc ttgcgtgcct tgaattactt ccacctggct
     3001 gcagtacgtg attcttgatc ccgagcttcg ggttggaagt gggtgggaga gttcgaggcc
     3061 ttgcgcttaa ggagcccctt cgcctcgtgc ttgagttgag gcctggcctg ggcgctgggg
     3121 ccgccgcgtg cgaatctggt ggcaccttcg cgcctgtctc gctgctttcg ataagtctct
     3181 agccatttaa aatttttgat gacctgctgc gacgcttttt ttctggcaag atagtcttgt
     3241 aaatgcgggc caagatctgc acactggtat ttcggttttt ggggccgcgg gcggcgacgg
     3301 ggcccgtgcg tcccagcgca catgttcggc gaggcggggc ctgcgagcgc ggccaccgag
     3361 aatcggacgg gggtagtctc aagctggccg gcctgctctg gtgcctggcc tcgcgccgcc
     3421 gtgtatcgcc ccgccctggg cggcaaggct ggcccggtcg gcaccagttg cgtgagcgga
     3481 aagatggccg cttcccggcc ctgctgcagg gagctcaaaa tggaggacgc ggcgctcggg
     3541 agagcgggcg ggtgagtcac ccacacaaag gaaaagggcc tttccgtcct cagccgtcgc
     3601 ttcatgtgac tccacggagt accgggcgcc gtccaggcac ctcgattagt tctcgagctt
     3661 ttggagtacg tcgtctttag gttgggggga ggggttttat gcgatggagt ttccccacac
     3721 tgagtgggtg gagactgaag ttaggccagc ttggcacttg atgtaattct ccttggaatt
     3781 tgcccttttt gagtttggat cttggttcat tctcaagcct cagacagtgg ttcaaagttt
     3841 ttttcttcca tttcaggtgt cgtgacgtac ggccaccatg aaaaggccgg cggccacgaa
     3901 aaaggccggc caggcaaaaa agaaaaagga caagaagtac agcatcggcc tggccatcgg
     3961 caccaactct gtgggctggg ccgtgatcac cgacgagtac aaggtgccca gcaagaaatt
     4021 caaggtgctg ggcaacaccg accggcacag catcaagaag aacctgatcg gagccctgct
     4081 gttcgacagc ggcgaaacag ccgaggccac ccggctgaag agaaccgcca gaagaagata
     4141 caccagacgg aagaaccgga tctgctatct gcaagagatc ttcagcaacg agatggccaa
     4201 ggtggacgac agcttcttcc acagactgga agagtccttc ctggtggaag aggataagaa
     4261 gcacgagcgg caccccatct tcggcaacat cgtggacgag gtggcctacc acgagaagta
     4321 ccccaccatc taccacctga gaaagaaact ggtggacagc accgacaagg ccgacctgcg
     4381 gctgatctat ctggccctgg cccacatgat caagttccgg ggccacttcc tgatcgaggg
     4441 cgacctgaac cccgacaaca gcgacgtgga caagctgttc atccagctgg tgcagaccta
     4501 caaccagctg ttcgaggaaa accccatcaa cgccagcggc gtggacgcca aggccatcct
     4561 gtctgccaga ctgagcaaga gcagacggct ggaaaatctg atcgcccagc tgcccggcga
     4621 gaagaagaat ggcctgttcg gcaacctgat tgccctgagc ctgggcctga cccccaactt
     4681 caagagcaac ttcgacctgg ccgaggatgc caaactgcag ctgagcaagg acacctacga
     4741 cgacgacctg gacaacctgc tggcccagat cggcgaccag tacgccgacc tgtttctggc
     4801 cgccaagaac ctgtccgacg ccatcctgct gagcgacatc ctgagagtga acaccgagat
     4861 caccaaggcc cccctgagcg cctctatgat caagagatac gacgagcacc accaggacct
     4921 gaccctgctg aaagctctcg tgcggcagca gctgcctgag aagtacaaag agattttctt
     4981 cgaccagagc aagaacggct acgccggcta cattgacggc ggagccagcc aggaagagtt
     5041 ctacaagttc atcaagccca tcctggaaaa gatggacggc accgaggaac tgctcgtgaa
     5101 gctgaacaga gaggacctgc tgcggaagca gcggaccttc gacaacggca gcatccccca
     5161 ccagatccac ctgggagagc tgcacgccat tctgcggcgg caggaagatt tttacccatt
     5221 cctgaaggac aaccgggaaa agatcgagaa gatcctgacc ttccgcatcc cctactacgt
     5281 gggccctctg gccaggggaa acagcagatt cgcctggatg accagaaaga gcgaggaaac
     5341 catcaccccc tggaacttcg aggaagtggt ggacaagggc gcttccgccc agagcttcat
     5401 cgagcggatg accaacttcg ataagaacct gcccaacgag aaggtgctgc ccaagcacag
     5461 cctgctgtac gagtacttca ccgtgtataa cgagctgacc aaagtgaaat acgtgaccga
     5521 gggaatgaga aagcccgcct tcctgagcgg cgagcagaaa aaggccatcg tggacctgct
     5581 gttcaagacc aaccggaaag tgaccgtgaa gcagctgaaa gaggactact tcaagaaaat
     5641 cgagtgcttc gactccgtgg aaatctccgg cgtggaagat cggttcaacg cctccctggg
     5701 cacataccac gatctgctga aaattatcaa ggacaaggac ttcctggaca atgaggaaaa
     5761 cgaggacatt ctggaagata tcgtgctgac cctgacactg tttgaggaca gagagatgat
     5821 cgaggaacgg ctgaaaacct atgcccacct gttcgacgac aaagtgatga agcagctgaa
     5881 gcggcggaga tacaccggct ggggcaggct gagccggaag ctgatcaacg gcatccggga
     5941 caagcagtcc ggcaagacaa tcctggattt cctgaagtcc gacggcttcg ccaacagaaa
     6001 cttcatgcag ctgatccacg acgacagcct gacctttaaa gaggacatcc agaaagccca
     6061 ggtgtccggc cagggcgata gcctgcacga gcacattgcc aatctggccg gcagccccgc
     6121 cattaagaag ggcatcctgc agacagtgaa ggtggtggac gagctcgtga aagtgatggg
     6181 ccggcacaag cccgagaaca tcgtgatcga aatggccaga gagaaccaga ccacccagaa
     6241 gggacagaag aacagccgcg agagaatgaa gcggatcgaa gagggcatca aagagctggg
     6301 cagccagatc ctgaaagaac accccgtgga aaacacccag ctgcagaacg agaagctgta
     6361 cctgtactac ctgcagaatg ggcgggatat gtacgtggac caggaactgg acatcaaccg
     6421 gctgtccgac tacgatgtgg accacatcgt gcctcagagc tttctgaagg acgactccat
     6481 cgacaacaag gtgctgacca gaagcgacaa ggcccggggc aagagcgaca acgtgccctc
     6541 cgaagaggtc gtgaagaaga tgaagaacta ctggcggcag ctgctgaacg ccaagctgat
     6601 tacccagaga aagttcgaca atctgaccaa ggccgagaga ggcggcctga gcgaactgga
     6661 taaggccggc ttcatcaaga gacagctggt ggaaacccgg cagatcacaa agcacgtggc
     6721 acagatcctg gactcccgga tgaacactaa gtacgacgag aatgacaagc tgatccggga
     6781 agtgaaagtg atcaccctga agtccaagct ggtgtccgat ttccggaagg atttccagtt
     6841 ttacaaagtg cgcgagatca acaactacca ccacgcccac gacgcctacc tgaacgccgt
     6901 cgtgggaacc gccctgatca aaaagtaccc taagctggaa agcgagttcg tgtacggcga
     6961 ctacaaggtg tacgacgtgc ggaagatgat cgccaagagc gagcaggaaa tcggcaaggc
     7021 taccgccaag tacttcttct acagcaacat catgaacttt ttcaagaccg agattaccct
     7081 ggccaacggc gagatccgga agcggcctct gatcgagaca aacggcgaaa ccggggagat
     7141 cgtgtgggat aagggccggg attttgccac cgtgcggaaa gtgctgagca tgccccaagt
     7201 gaatatcgtg aaaaagaccg aggtgcagac aggcggcttc agcaaagagt ctatcctgcc
     7261 caagaggaac agcgataagc tgatcgccag aaagaaggac tgggacccta agaagtacgg
     7321 cggcttcgac agccccaccg tggcctattc tgtgctggtg gtggccaaag tggaaaaggg
     7381 caagtccaag aaactgaaga gtgtgaaaga gctgctgggg atcaccatca tggaaagaag
     7441 cagcttcgag aagaatccca tcgactttct ggaagccaag ggctacaaag aagtgaaaaa
     7501 ggacctgatc atcaagctgc ctaagtactc cctgttcgag ctggaaaacg gccggaagag
     7561 aatgctggcc tctgccggcg aactgcagaa gggaaacgaa ctggccctgc cctccaaata
     7621 tgtgaacttc ctgtacctgg ccagccacta tgagaagctg aagggctccc ccgaggataa
     7681 tgagcagaaa cagctgtttg tggaacagca caagcactac ctggacgaga tcatcgagca
     7741 gatcagcgag ttctccaaga gagtgatcct ggccgacgct aatctggaca aagtgctgtc
     7801 cgcctacaac aagcaccggg ataagcccat cagagagcag gccgagaata tcatccacct
     7861 gtttaccctg accaatctgg gagcccctgc cgccttcaag tactttgaca ccaccatcga
     7921 ccggaagagg tacaccagca ccaaagaggt gctggacgcc accctgatcc accagagcat
     7981 caccggcctg tacgagacac ggatcgacct gtctcagctg ggaggcgaca gcgctggagg
     8041 aggtggaagc ggaggaggag gaagcggagg aggaggtagc ggacctaaga aaaagaggaa
     8101 ggtggcggcc gctggatcca ttttcaaacc agaagaacta cgacaggcac tgatgccaac
     8161 tttggaggca ctttaccgtc aggacccaga atcccttccc tttcgtcaac ctgtggaccc
     8221 tcagctttta ggaatccctg attactttga tattgtgaag agccccatgg atctttctac
     8281 cattaagagg aagttagaca ctggacagta tcaggagccc tggcagtatg tcgatgatat
     8341 ttggcttatg ttcaataatg cctggttata taaccggaaa acatcacggg tatacaaata
     8401 ctgctccaag ctctctgagg tctttgaaca agaaattgac ccagtgatgc aaagccttgg
     8461 atactgttgt ggcagaaagt tggagttctc tccacagaca ctgtgttgct acggcaaaca
     8521 gttgtgcaca atacctcgtg atgccactta ttacagttac cagaacaggt atcatttctg
     8581 tgagaagtgt ttcaatgaga tccaagggga gagcgtttct ttgggggatg acccttccca
     8641 gcctcaaact acaataaata aagaacaatt ttccaagaga aaaaatgaca cactggaccc
     8701 tgaactgttt gttgaatgta cggagtgcgg aagaaagatg catcagatct gtgtccttca
     8761 ccatgagatc atctggcctg ctggattcgt ctgtgatggc tgtttaaaga aaagtgcacg
     8821 aactaggaaa gaaaataagt tttctgctaa aaggttgcca tctaccagac ttggcacctt
     8881 tctagagaat cgtgtgaatg actttctgag gcgacagaat caccctgagt caggagaggt
     8941 cactgttaga gtagttcatg cttctgacaa aaccgtggaa gtaaaaccag gcatgaaagc
     9001 aaggtttgtg gacagtggag agatggcaga atcctttcca taccgaacca aagccctctt
     9061 tgcctttgaa gaaattgatg gtgttgacct gtgcttcttt ggcatgcatg ttcaagagta
     9121 tggctctgac tgccctccac ccaaccagag gagagtatac atatcttacc tcgatagtgt
     9181 tcatttcttc cgtcctaaat gcttgaggac tgcagtctat catgaaatcc taattggata
     9241 tttagaatat gtcaagaaat taggttacac aacagggcat atttgggcat gtccaccaag
     9301 tgagggagat gattatatct tccattgcca tcctcctgac cagaagatac ccaagcccaa
     9361 gcgactgcag gaatggtaca aaaaaatgct tgacaaggct gtatcagagc gtattgtcca
     9421 tgactacaag gatattttta aacaagctac tgaagataga ttaacaagtg caaaggaatt
     9481 gccttatttc gagggtgatt tctggcccaa tgttctggaa gaaagcatta aggaactgga
     9541 acaggaggaa gaagagagaa aacgagagga aaacaccagc aatgaaagca cagatgtgac
     9601 caagggagac agcaaaaatg ctaaaaagaa gaataataag aaaaccagca aaaataagag
     9661 cagcctgagt aggggcaaca agaagaaacc cgggatgccc aatgtatcta acgacctctc
     9721 acagaaacta tatgccacca tggagaagca taaagaggtc ttctttgtga tccgcctcat
     9781 tgctggccct gctgccaact ccctgcctcc cattgttgat cctgatcctc tcatcccctg
     9841 cgatctgatg gatggtcggg atgcgtttct cacgctggca agggacaagc acctggagtt
     9901 ctcttcactc cgaagagccc agtggtccac catgtgcatg ctggtggagc tgcacacgca
     9961 gagccaggac tacccgtacg acgttccgga ctacgcttct gatatctgta caggcagtgg
    10021 agagggcaga ggaagtctgc taacatgcgg tgacgtcgag gagaatcctg gcccaatggc
    10081 caagcctttg tctcaagaag aatccaccct cattgaaaga gcaacggcta caatcaacag
    10141 catccccatc tctgaagact acagcgtcgc cagcgcagct ctctctagcg acggccgcat
    10201 cttcactggt gtcaatgtat atcattttac tgggggacct tgtgcagaac tcgtggtgct
    10261 gggcactgct gctgctgcgg cagctggcaa cctgacttgt atcgtcgcga tcggaaatga
    10321 gaacaggggc atcttgagcc cctgcggacg gtgccgacag gtgcttctcg atctgcatcc
    10381 tgggatcaaa gccatagtga aggacagtga tggacagccg acggcagttg ggattcgtga
    10441 attgctgccc tctggttatg tgtgggaggg ctaagaattc gatatcaagc ttatcggtaa
    10501 tcaacctctg gattacaaaa tttgtgaaag attgactggt attcttaact atgttgctcc
    10561 ttttacgcta tgtggatacg ctgctttaat gcctttgtat catgctattg cttcccgtat
    10621 ggctttcatt ttctcctcct tgtataaatc ctggttgctg tctctttatg aggagttgtg
    10681 gcccgttgtc aggcaacgtg gcgtggtgtg cactgtgttt gctgacgcaa cccccactgg
    10741 ttggggcatt gccaccacct gtcagctcct ttccgggact ttcgctttcc ccctccctat
    10801 tgccacggcg gaactcatcg ccgcctgcct tgcccgctgc tggacagggg ctcggctgtt
    10861 gggcactgac aattccgtgg tgttgtcggg gaaatcatcg tcctttcctt ggctgctcgc
    10921 ctgtgttgcc acctggattc tgcgcgggac gtccttctgc tacgtccctt cggccctcaa
    10981 tccagcggac cttccttccc gcggcctgct gccggctctg cggcctcttc cgcgtcttcg
    11041 ccttcgccct cagacgagtc ggatctccct ttgggccgcc tccccgcatc gataccgtcg
    11101 acctcgagac ctagaaaaac atggagcaat cacaagtagc aatacagcag ctaccaatgc
    11161 tgattgtgcc tggctagaag cacaagagga ggaggaggtg ggttttccag tcacacctca
    11221 ggtaccttta agaccaatga cttacaaggc agctgtagat cttagccact ttttaaaaga
    11281 aaagggggga ctggaagggc taattcactc ccaacgaaga caagatatcc ttgatctgtg
    11341 gatctaccac acacaaggct acttccctga ttggcagaac tacacaccag ggccagggat
    11401 cagatatcca ctgacctttg gatggtgcta caagctagta ccagttgagc aagagaaggt
    11461 agaagaagcc aatgaaggag agaacacccg cttgttacac cctgtgagcc tgcatgggat
    11521 ggatgacccg gagagagaag tattagagtg gaggtttgac agccgcctag catttcatca
    11581 catggcccga gagctgcatc cggactgtac tgggtctctc tggttagacc agatctgagc
    11641 ctgggagctc tctggctaac tagggaaccc actgcttaag cctcaataaa gcttgccttg
    11701 agtgcttcaa gtagtgtgtg cccgtctgtt gtgtgactct ggtaactaga gatccctcag
    11761 acccttttag tcagtgtgga aaatctctag cagggcccgt ttaaacccgc tgatcagcct
    11821 cgactgtgcc ttctagttgc cagccatctg ttgtttgccc ctcccccgtg ccttccttga
    11881 ccctggaagg tgccactccc actgtccttt cctaataaaa tgaggaaatt gcatcgcatt
    11941 gtctgagtag gtgtcattct attctggggg gtggggtggg gcaggacagc aagggggagg
    12001 attgggaaga caatagcagg catgctgggg atgcggtggg ctctatggct tctgaggcgg
    12061 aaagaaccag ctggggctct agggggtatc cccacgcgcc ctgtagcggc gcattaagcg
    12121 cggcgggtgt ggtggttacg cgcagcgtga ccgctacact tgccagcgcc ctagcgcccg
    12181 ctcctttcgc tttcttccct tcctttctcg ccacgttcgc cggctttccc cgtcaagctc
    12241 taaatcgggg gctcccttta gggttccgat ttagtgcttt acggcacctc gaccccaaaa
    12301 aacttgatta gggtgatggt tcacgtagtg ggccatcgcc ctgatagacg gtttttcgcc
    12361 ctttgacgtt ggagtccacg ttctttaata gtggactctt gttccaaact ggaacaacac
    12421 tcaaccctat ctcggtctat tcttttgatt tataagggat tttgccgatt tcggcctatt
    12481 ggttaaaaaa tgagctgatt taacaaaaat ttaacgcgaa ttaattctgt ggaatgtgtg
    12541 tcagttaggg tgtggaaagt ccccaggctc cccagcaggc agaagtatgc aaagcatgca
    12601 tctcaattag tcagcaacca ggtgtggaaa gtccccaggc tccccagcag gcagaagtat
    12661 gcaaagcatg catctcaatt agtcagcaac catagtcccg cccctaactc cgcccatccc
    12721 gcccctaact ccgcccagtt ccgcccattc tccgccccat ggctgactaa ttttttttat
    12781 ttatgcagag gccgaggccg cctctgcctc tgagctattc cagaagtagt gaggaggctt
    12841 ttttggaggc ctaggctttt gcaaaaagct cccgggagct tgtatatcca ttttcggatc
    12901 tgatcagcac gtgttgacaa ttaatcatcg gcatagtata tcggcatagt ataatacgac
    12961 aaggtgagga actaaaccat ggccaagttg accagtgccg ttccggtgct caccgcgcgc
    13021 gacgtcgccg gagcggtcga gttctggacc gaccggctcg ggttctcccg ggacttcgtg
    13081 gaggacgact tcgccggtgt ggtccgggac gacgtgaccc tgttcatcag cgcggtccag
    13141 gaccaggtgg tgccggacaa caccctggcc tgggtgtggg tgcgcggcct ggacgagctg
    13201 tacgccgagt ggtcggaggt cgtgtccacg aacttccggg acgcctccgg gccggccatg
    13261 accgagatcg gcgagcagcc gtgggggcgg gagttcgccc tgcgcgaccc ggccggcaac
    13321 tgcgtgcact tcgtggccga ggagcaggac tgacacgtgc tacgagattt cgattccacc
    13381 gccgccttct atgaaaggtt gggcttcgga atcgttttcc gggacgccgg ctggatgatc
    13441 ctccagcgcg gggatctcat gctggagttc ttcgcccacc ccaacttgtt tattgcagct
    13501 tataatggtt acaaataaag caatagcatc acaaatttca caaataaagc atttttttca
    13561 ctgcattcta gttgtggttt gtccaaactc atcaatgtat cttatcatgt ctgtataccg
    13621 tcgacctcta gctagagctt ggcgtaatca tggtcatagc tgtttcctgt gtgaaattgt
    13681 tatccgctca caattccaca caacatacga gccggaagca taaagtgtaa agcctggggt
    13741 gcctaatgag tgagctaact cacattaatt gcgttgcgct cactgcccgc tttccagtcg
    13801 ggaaacctgt cgtgccagct gcattaatga atcggccaac gcgcggggag aggcggtttg
    13861 cgtattgggc gctcttccgc ttcctcgctc actgactcgc tgcgctcggt cgttcggctg
    13921 cggcgagcgg tatcagctca ctcaaaggcg gtaatacggt tatccacaga atcaggggat
    13981 aacgcaggaa agaacatgtg agcaaaaggc cagcaaaagg ccaggaaccg taaaaaggcc
    14041 gcgttgctgg cgtttttcca taggctccgc ccccctgacg agcatcacaa aaatcgacgc
    14101 tcaagtcaga ggtggcgaaa cccgacagga ctataaagat accaggcgtt tccccctgga
    14161 agctccctcg tgcgctctcc tgttccgacc ctgccgctta ccggatacct gtccgccttt
    14221 ctcccttcgg gaagcgtggc gctttctcat agctcacgct gtaggtatct cagttcggtg
    14281 taggtcgttc gctccaagct gggctgtgtg cacgaacccc ccgttcagcc cgaccgctgc
    14341 gccttatccg gtaactatcg tcttgagtcc aacccggtaa gacacgactt atcgccactg
    14401 gcagcagcca ctggtaacag gattagcaga gcgaggtatg taggcggtgc tacagagttc
    14461 ttgaagtggt ggcctaacta cggctacact agaagaacag tatttggtat ctgcgctctg
    14521 ctgaagccag ttaccttcgg aaaaagagtt ggtagctctt gatccggcaa acaaaccacc
    14581 gctggtagcg gtggtttttt tgtttgcaag cagcagatta cgcgcagaaa aaaaggatct
    14641 caagaagatc ctttgatctt ttctacgggg tctgacgctc agtggaacga aaactcacgt
    14701 taagggattt tggtcatgag attatcaaaa aggatcttca cctagatcct tttaaattaa
    14761 aaatgaagtt ttaaatcaat ctaaagtata tatgagtaaa cttggtctga cagttaccaa
    14821 tgcttaatca gtgaggcacc tatctcagcg atctgtctat ttcgttcatc catagttgcc
    14881 tgactccccg tcgtgtagat aactacgata cgggagggct taccatctgg ccccagtgct
    14941 gcaatgatac cgcgagaccc acgctcaccg gctccagatt tatcagcaat aaaccagcca
    15001 gccggaaggg ccgagcgcag aagtggtcct gcaactttat ccgcctccat ccagtctatt
    15061 aattgttgcc gggaagctag agtaagtagt tcgccagtta atagtttgcg caacgttgtt
    15121 gccattgcta caggcatcgt ggtgtcacgc tcgtcgtttg gtatggcttc attcagctcc
    15181 ggttcccaac gatcaaggcg agttacatga tcccccatgt tgtgcaaaaa agcggttagc
    15241 tccttcggtc ctccgatcgt tgtcagaagt aagttggccg cagtgttatc actcatggtt
    15301 atggcagcac tgcataattc tcttactgtc atgccatccg taagatgctt ttctgtgact
    15361 ggtgagtact caaccaagtc attctgagaa tagtgtatgc ggcgaccgag ttgctcttgc
    15421 ccggcgtcaa tacgggataa taccgcgcca catagcagaa ctttaaaagt gctcatcatt
    15481 ggaaaacgtt cttcggggcg aaaactctca aggatcttac cgctgttgag atccagttcg
    15541 atgtaaccca ctcgtgcacc caactgatct tcagcatctt ttactttcac cagcgtttct
    15601 gggtgagcaa aaacaggaag gcaaaatgcc gcaaaaaagg gaataagggc gacacggaaa
    15661 tgttgaatac tcatactctt cctttttcaa tattattgaa gcatttatca gggttattgt
    15721 ctcatgagcg gatacatatt tgaatgtatt tagaaaaata aacaaatagg ggttccgcgc
    15781 acatttcccc gaaaagtgcc acctgac
//
