LOCUS       sequence-324509-       14290 bp    DNA     circular SYN 15-DEC-2025
DEFINITION  Lentiviral expression of dSpCas9-2xVP64 with blasticidin resistance.
ACCESSION   .
VERSION     .
KEYWORDS    .
SOURCE      synthetic DNA construct
  ORGANISM  synthetic DNA construct
REFERENCE   1  (bases 1 to 14290)
  AUTHORS   Black JB, McCutcheon SR, Dube S, Barrera A, Klann TS, Rice GA, Adkar
            SS, Soderling SH, Reddy TE, Gersbach CA
  TITLE     Master Regulators and Cofactors of Human Neuronal Cell Fate 
            Specification Identified by CRISPR Gene Activation Screens.
  JOURNAL   Cell Rep. 2020 Dec 1;33(9):108460. doi: 
            10.1016/j.celrep.2020.108460.
  PUBMED    33264623
REFERENCE   2  (bases 1 to 14290)
  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: "Cell Rep.";
            date: "2020-12-1"; volume: "33(9):108460. doi"; pages: " 
            10.1016/j.celrep.2020.108460"
COMMENT     Sequence Label: pLV_hUbC-dSpCas9-2xVP64-T2A-BSD
FEATURES             Location/Qualifiers
     source          1..14290
                     /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        2622..3833
                     /label=UbC promoter
                     /note="human ubiquitin C promoter"
     primer_bind     3670..3689
                     /label=hUBCpro-F
                     /note="Human Ubiquitin C (UbC) promoter, forward primer"
     regulatory      3862..3871
                     /label=Kozak sequence
                     /note="vertebrate consensus sequence for strong initiation 
                     of translation (Kozak, 1987)"
                     /regulatory_class="other"
     CDS             3871..3936
                     /codon_start=1
                     /product="three tandem FLAG(R) epitope tags, followed by an
                     enterokinase cleavage site"
                     /label=3xFLAG
                     /translation="DYKDHDGDYKDHDIDYKDDDDK"
     CDS             3952..4098
                     /codon_start=1
                     /product="tetrameric repeat of the minimal activation 
                     domain of herpes simplex virus VP16 (Beerli et al., 1998)"
                     /label=VP64
                     /translation="DALDDFDLDMLGSDALDDFDLDMLGSDALDDFDLDMLGSDALDDF
                     DLDM"
     CDS             4105..4125
                     /codon_start=1
                     /product="nuclear localization signal of SV40 (simian virus
                     40) large T antigen"
                     /label=SV40 NLS
                     /translation="PKKKRKV"
     CDS             join(4135..4161,4162..4164,4165..6648,6649..6654,6655..6720,
                     6721..6723,6724..8238)
                     /codon_start=1
                     /product="catalytically dead mutant of the Cas9 
                     endonuclease from the Streptococcus pyogenes Type II 
                     CRISPR/Cas system"
                     /label=Cas9m4
                     /note="RNA-guided DNA-binding protein that lacks 
                     endonuclease activity due to the mutations D10A, D839A, 
                     H840A, and N863A (Mali et al., 2013)"
                     /translation="MDKKYSIGLAIGTNSVGWAVITDEYKVPSKKFKVLGNTDRHSIKK
                     NLIGALLFDSGETAEATRLKRTARRRYTRRKNRICYLQEIFSNEMAKVDDSFFHRLEES
                     FLVEEDKKHERHPIFGNIVDEVAYHEKYPTIYHLRKKLVDSTDKADLRLIYLALAHMIK
                     FRGHFLIEGDLNPDNSDVDKLFIQLVQTYNQLFEENPINASGVDAKAILSARLSKSRRL
                     ENLIAQLPGEKKNGLFGNLIALSLGLTPNFKSNFDLAEDAKLQLSKDTYDDDLDNLLAQ
                     IGDQYADLFLAAKNLSDAILLSDILRVNTEITKAPLSASMIKRYDEHHQDLTLLKALVR
                     QQLPEKYKEIFFDQSKNGYAGYIDGGASQEEFYKFIKPILEKMDGTEELLVKLNREDLL
                     RKQRTFDNGSIPHQIHLGELHAILRRQEDFYPFLKDNREKIEKILTFRIPYYVGPLARG
                     NSRFAWMTRKSEETITPWNFEEVVDKGASAQSFIERMTNFDKNLPNEKVLPKHSLLYEY
                     FTVYNELTKVKYVTEGMRKPAFLSGEQKKAIVDLLFKTNRKVTVKQLKEDYFKKIECFD
                     SVEISGVEDRFNASLGTYHDLLKIIKDKDFLDNEENEDILEDIVLTLTLFEDREMIEER
                     LKTYAHLFDDKVMKQLKRRRYTGWGRLSRKLINGIRDKQSGKTILDFLKSDGFANRNFM
                     QLIHDDSLTFKEDIQKAQVSGQGDSLHEHIANLAGSPAIKKGILQTVKVVDELVKVMGR
                     HKPENIVIEMARENQTTQKGQKNSRERMKRIEEGIKELGSQILKEHPVENTQLQNEKLY
                     LYYLQNGRDMYVDQELDINRLSDYDVDAIVPQSFLKDDSIDNKVLTRSDKNRGKSDNVP
                     SEEVVKKMKNYWRQLLNAKLITQRKFDNLTKAERGGLSELDKAGFIKRQLVETRQITKH
                     VAQILDSRMNTKYDENDKLIREVKVITLKSKLVSDFRKDFQFYKVREINNYHHAHDAYL
                     NAVVGTALIKKYPKLESEFVYGDYKVYDVRKMIAKSEQEIGKATAKYFFYSNIMNFFKT
                     EITLANGEIRKRPLIETNGETGEIVWDKGRDFATVRKVLSMPQVNIVKKTEVQTGGFSK
                     ESILPKRNSDKLIARKKDWDPKKYGGFDSPTVAYSVLVVAKVEKGKSKKLKSVKELLGI
                     TIMERSSFEKNPIDFLEAKGYKEVKKDLIIKLPKYSLFELENGRKRMLASAGELQKGNE
                     LALPSKYVNFLYLASHYEKLKGSPEDNEQKQLFVEQHKHYLDEIIEQISEFSKRVILAD
                     ANLDKVLSAYNKHRDKPIREQAENIIHLFTLTNLGAPAAFKYFDTTIDRKRYTSTKEVL
                     DATLIHQSITGLYETRIDLSQLGGD"
     CDS             join(4135..4161,4162..4164,4165..6651,6652..6654,
                     6655..8238)
                     /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             4135..8238
                     /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             8251..8271
                     /codon_start=1
                     /product="nuclear localization signal of SV40 (simian virus
                     40) large T antigen"
                     /label=SV40 NLS
                     /translation="PKKKRKV"
     CDS             8284..8433
                     /codon_start=1
                     /product="tetrameric repeat of the minimal activation 
                     domain of herpes simplex virus VP16 (Beerli et al., 1998)"
                     /label=VP64
                     /translation="DALDDFDLDMLGSDALDDFDLDMLGSDALDDFDLDMLGSDALDDF
                     DLDML"
     CDS             8443..8496
                     /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             8503..8898
                     /codon_start=1
                     /gene="Aspergillus terreus BSD"
                     /product="blasticidin S deaminase"
                     /label=BSD
                     /note="confers resistance to blasticidin"
                     /translation="MAKPLSQEESTLIERATATINSIPISEDYSVASAALSSDGRIFTG
                     VNVYHFTGGPCAELVVLGTAAAAAAGNLTCIVAIGNENRGILSPCGRCRQVLLDLHPGI
                     KAIVKDSDGQPTAVGIRELLPSGYVWEG"
     protein_bind    complement(8924..8957)
                     /label=loxP
                     /bound_moiety="Cre recombinase"
                     /note="Cre-mediated recombination occurs in the 8-bp core 
                     sequence (ATGTATGC) (Shaw et al., 2021)."
     misc_feature    8982..9570
                     /label=WPRE
                     /note="woodchuck hepatitis virus posttranscriptional 
                     regulatory element"
     primer_bind     complement(9035..9055)
                     /label=WPRE-R
                     /note="WPRE, reverse primer"
     CDS             complement(9453..9464)
                     /codon_start=1
                     /product="Factor Xa recognition and cleavage site"
                     /label=Factor Xa site
                     /translation="IEGR"
     primer_bind     complement(9573..9589)
                     /label=KS primer
                     /note="common sequencing primer, one of multiple similar 
                     variants"
     primer_bind     complement(9574..9590)
                     /label=pBluescriptKS
                     /note="For pBluescript vector"
     LTR             10095..10275
                     /label=5' LTR (truncated)
                     /note="truncated 5' long terminal repeat (LTR) from HIV-1"
     primer_bind     complement(10301..10318)
                     /label=BGH-rev
                     /note="Bovine growth hormone terminator, reverse primer. 
                     Also called BGH reverse"
     polyA_signal    10307..10531
                     /label=bGH poly(A) signal
                     /note="bovine growth hormone polyadenylation signal"
     rep_origin      10577..11005
                     /direction=RIGHT
                     /label=f1 ori
                     /note="f1 bacteriophage origin of replication; arrow 
                     indicates direction of (+) strand synthesis"
     primer_bind     complement(10664..10683)
                     /label=F1ori-R
                     /note="F1 origin, reverse primer"
     primer_bind     10874..10895
                     /label=F1ori-F
                     /note="F1 origin, forward primer"
     primer_bind     complement(11014..11034)
                     /label=pBABE 3'
                     /note="SV40 enhancer, reverse primer for pBABE vectors"
     promoter        11019..11348
                     /label=SV40 promoter
                     /note="SV40 enhancer and early promoter"
     rep_origin      11199..11334
                     /label=SV40 ori
                     /note="SV40 origin of replication"
     primer_bind     11261..11280
                     /label=SV40pro-F
                     /note="SV40 promoter/origin, forward primer"
     promoter        11396..11443
                     /label=EM7 promoter
                     /note="synthetic bacterial promoter "
     CDS             11462..11836
                     /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    11966..12087
                     /label=SV40 poly(A) signal
                     /note="SV40 polyadenylation signal"
     primer_bind     complement(12003..12022)
                     /label=SV40pA-R
                     /note="SV40 polyA, reverse primer"
     primer_bind     12057..12076
                     /label=EBV-rev
                     /note="SV40 polyA terminator, reverse primer"
     primer_bind     complement(12136..12152)
                     /label=M13 rev
                     /note="common sequencing primer, one of multiple similar 
                     variants"
     primer_bind     complement(12136..12152)
                     /label=M13 Reverse
                     /note="In lacZ gene. Also called M13-rev"
     primer_bind     complement(12149..12171)
                     /label=M13/pUC Reverse
                     /note="In lacZ gene"
     protein_bind    12160..12176
                     /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(12184..12190,12191..12208,12209..12214))
                     /label=lac promoter
                     /note="promoter for the E. coli lac operon"
     protein_bind    12229..12250
                     /label=CAP binding site
                     /bound_moiety="E. coli catabolite activator protein"
                     /note="CAP binding activates transcription in the presence 
                     of cAMP."
     primer_bind     complement(12367..12384)
                     /label=L4440
                     /note="L4440 vector, forward primer"
     rep_origin      complement(12538..13126)
                     /direction=LEFT
                     /label=ori
                     /note="high-copy-number ColE1/pMB1/pBR322/pUC origin of 
                     replication"
     primer_bind     complement(12618..12637)
                     /label=pBR322ori-F
                     /note="pBR322 origin, forward primer"
     CDS             complement(join(13297..14088,14089..14157))
                     /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     13920..13939
                     /label=Amp-R
                     /note="Ampicillin resistance gene, reverse primer"
     promoter        complement(14158..14262)
                     /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 acccgtgtcg gctccagatc tggcctccgc gccgggtttt
     2641 ggcgcctccc gcgggcgccc ccctcctcac ggcgagcgct gccacgtcag acgaagggcg
     2701 cagcgagcgt cctgatcctt ccgcccggac gctcaggaca gcggcccgct gctcataaga
     2761 ctcggcctta gaaccccagt atcagcagaa ggacatttta ggacgggact tgggtgactc
     2821 tagggcactg gttttctttc cagagagcgg aacaggcgag gaaaagtagt cccttctcgg
     2881 cgattctgcg gagggatctc cgtggggcgg tgaacgccga tgattatata aggacgcgcc
     2941 gggtgtggca cagctagttc cgtcgcagcc gggatttggg tcgcggttct tgtttgtgga
     3001 tcgctgtgat cgtcacttgg tgagtagcgg gctgctgggc tggccggggc tttcgtggcc
     3061 gccgggccgc tcggtgggac ggaagcgtgt ggagagaccg ccaagggctg tagtctgggt
     3121 ccgcgagcaa ggttgccctg aactgggggt tggggggagc gcagcaaaat ggcggctgtt
     3181 cccgagtctt gaatggaaga cgcttgtgag gcgggctgtg aggtcgttga aacaaggtgg
     3241 ggggcatggt gggcggcaag aacccaaggt cttgaggcct tcgctaatgc gggaaagctc
     3301 ttattcgggt gagatgggct ggggcaccat ctggggaccc tgacgtgaag tttgtcactg
     3361 actggagaac tcggtttgtc gtctgttgcg ggggcggcag ttatggcggt gccgttgggc
     3421 agtgcacccg tacctttggg agcgcgcgcc ctcgtcgtgt cgtgacgtca cccgttctgt
     3481 tggcttataa tgcagggtgg ggccacctgc cggtaggtgt gcggtaggct tttctccgtc
     3541 gcaggacgca gggttcgggc ctagggtagg ctctcctgaa tcgacaggcg ccggacctct
     3601 ggtgagggga gggataagtg aggcgtcagt ttctttggtc ggttttatgt acctatcttc
     3661 ttaagtagct gaagctccgg ttttgaacta tgcgctcggg gttggcgagt gtgttttgtg
     3721 aagtttttta ggcacctttt gaaatgtaat catttgggtc aatatgtaat tttcagtgtt
     3781 agactagtaa attgtccgct aaattctggc cgtttttggc ttttttgtta gacgaagctt
     3841 gggctgcagg tcgactctag agccaccatg gactacaaag accatgacgg tgattataaa
     3901 gatcatgaca tcgattacaa ggatgacgat gacaagcacg gtccgcgggc tgacgcattg
     3961 gacgattttg atctggatat gctgggaagt gacgccctcg atgattttga ccttgacatg
     4021 cttggttcgg atgcccttga tgactttgac ctcgacatgc tcggcagtga cgcccttgat
     4081 gatttcgacc tggacatggt taaccccaag aagaagagga aggtgggccg cggaatggac
     4141 aagaagtact ccattgggct cgccatcggc acaaacagcg tcggctgggc cgtcattacg
     4201 gacgagtaca aggtgccgag caaaaaattc aaagttctgg gcaataccga tcgccacagc
     4261 ataaagaaga acctcattgg cgccctcctg ttcgactccg gggaaaccgc cgaagccacg
     4321 cggctcaaaa gaacagcacg gcgcagatat acccgcagaa agaatcggat ctgctacctg
     4381 caggagatct ttagtaatga gatggctaag gtggatgact ctttcttcca taggctggag
     4441 gagtcctttt tggtggagga ggataaaaag cacgagcgcc acccaatctt tggcaatatc
     4501 gtggacgagg tggcgtacca tgaaaagtac ccaaccatat atcatctgag gaagaagctt
     4561 gtagacagta ctgataaggc tgacttgcgg ttgatctatc tcgcgctggc gcatatgatc
     4621 aaatttcggg gacacttcct catcgagggg gacctgaacc cagacaacag cgatgtcgac
     4681 aaactcttta tccaactggt tcagacttac aatcagcttt tcgaagagaa cccgatcaac
     4741 gcatccggag ttgacgccaa agcaatcctg agcgctaggc tgtccaaatc ccggcggctc
     4801 gaaaacctca tcgcacagct ccctggggag aagaagaacg gcctgtttgg taatcttatc
     4861 gccctgtcac tcgggctgac ccccaacttt aaatctaact tcgacctggc cgaagatgcc
     4921 aagcttcaac tgagcaaaga cacctacgat gatgatctcg acaatctgct ggcccagatc
     4981 ggcgaccagt acgcagacct ttttttggcg gcaaagaacc tgtcagacgc cattctgctg
     5041 agtgatattc tgcgagtgaa cacggagatc accaaagctc cgctgagcgc tagtatgatc
     5101 aagcgctatg atgagcacca ccaagacttg actttgctga aggcccttgt cagacagcaa
     5161 ctgcctgaga agtacaagga aattttcttc gatcagtcta aaaatggcta cgccggatac
     5221 attgacggcg gagcaagcca ggaggaattt tacaaattta ttaagcccat cttggaaaaa
     5281 atggacggca ccgaggagct gctggtaaag cttaacagag aagatctgtt gcgcaaacag
     5341 cgcactttcg acaatggaag catcccccac cagattcacc tgggcgaact gcacgctatc
     5401 ctcaggcggc aagaggattt ctaccccttt ttgaaagata acagggaaaa gattgagaaa
     5461 atcctcacat ttcggatacc ctactatgta ggccccctcg cccggggaaa ttccagattc
     5521 gcgtggatga ctcgcaaatc agaagagacc atcactccct ggaacttcga ggaagtcgtg
     5581 gataaggggg cctctgccca gtccttcatc gaaaggatga ctaactttga taaaaatctg
     5641 cctaacgaaa aggtgcttcc taaacactct ctgctgtacg agtacttcac agtttataac
     5701 gagctcacca aggtcaaata cgtcacagaa gggatgagaa agccagcatt cctgtctgga
     5761 gagcagaaga aagctatcgt ggacctcctc ttcaagacga accggaaagt taccgtgaaa
     5821 cagctcaaag aagactattt caaaaagatt gaatgtttcg actctgttga aatcagcgga
     5881 gtggaggatc gcttcaacgc atccctggga acgtatcacg atctcctgaa aatcattaaa
     5941 gacaaggact tcctggacaa tgaggagaac gaggacattc ttgaggacat tgtcctcacc
     6001 cttacgttgt ttgaagatag ggagatgatt gaagaacgct tgaaaactta cgctcatctc
     6061 ttcgacgaca aagtcatgaa acagctcaag aggcgccgat atacaggatg ggggcggctg
     6121 tcaagaaaac tgatcaatgg gatccgagac aagcagagtg gaaagacaat cctggatttt
     6181 cttaagtccg atggatttgc caaccggaac ttcatgcagt tgatccatga tgactctctc
     6241 acctttaagg aggacatcca gaaagcacaa gtttctggcc agggggacag tcttcacgag
     6301 cacatcgcta atcttgcagg tagcccagct atcaaaaagg gaatactgca gaccgttaag
     6361 gtcgtggatg aactcgtcaa agtaatggga aggcataagc ccgagaatat cgttatcgag
     6421 atggcccgag agaaccaaac tacccagaag ggacagaaga acagtaggga aaggatgaag
     6481 aggattgaag agggtataaa agaactgggg tcccaaatcc ttaaggaaca cccagttgaa
     6541 aacacccagc ttcagaatga gaagctctac ctgtactacc tgcagaacgg cagggacatg
     6601 tacgtggatc aggaactgga catcaatcgg ctctccgact acgacgtgga tgccatcgtg
     6661 ccccagtctt ttctcaaaga tgattctatt gataataaag tgttgacaag atccgataaa
     6721 aatagaggga agagtgataa cgtcccctca gaagaagttg tcaagaaaat gaaaaattat
     6781 tggcggcagc tgctgaacgc caaactgatc acacaacgga agttcgataa tctgactaag
     6841 gctgaacgag gtggcctgtc tgagttggat aaagccggct tcatcaaaag gcagcttgtt
     6901 gagacacgcc agatcaccaa gcacgtggcc caaattctcg attcacgcat gaacaccaag
     6961 tacgatgaaa atgacaaact gattcgagag gtgaaagtta ttactctgaa gtctaagctg
     7021 gtctcagatt tcagaaagga ctttcagttt tataaggtga gagagatcaa caattaccac
     7081 catgcgcatg atgcctacct gaatgcagtg gtaggcactg cacttatcaa aaaatatccc
     7141 aagcttgaat ctgaatttgt ttacggagac tataaagtgt acgatgttag gaaaatgatc
     7201 gcaaagtctg agcaggaaat aggcaaggcc accgctaagt acttctttta cagcaatatt
     7261 atgaattttt tcaagaccga gattacactg gccaatggag agattcggaa gcgaccactt
     7321 atcgaaacaa acggagaaac aggagaaatc gtgtgggaca agggtaggga tttcgcgaca
     7381 gtccggaagg tcctgtccat gccgcaggtg aacatcgtta aaaagaccga agtacagacc
     7441 ggaggcttct ccaaggaaag tatcctcccg aaaaggaaca gcgacaagct gatcgcacgc
     7501 aaaaaagatt gggaccccaa gaaatacggc ggattcgatt ctcctacagt cgcttacagt
     7561 gtactggttg tggccaaagt ggagaaaggg aagtctaaaa aactcaaaag cgtcaaggaa
     7621 ctgctgggca tcacaatcat ggagcgatca agcttcgaaa aaaaccccat cgactttctc
     7681 gaggcgaaag gatataaaga ggtcaaaaaa gacctcatca ttaagcttcc caagtactct
     7741 ctctttgagc ttgaaaacgg ccggaaacga atgctcgcta gtgcgggcga gctgcagaaa
     7801 ggtaacgagc tggcactgcc ctctaaatac gttaatttct tgtatctggc cagccactat
     7861 gaaaagctca aagggtctcc cgaagataat gagcagaagc agctgttcgt ggaacaacac
     7921 aaacactacc ttgatgagat catcgagcaa ataagcgaat tctccaaaag agtgatcctc
     7981 gccgacgcta acctcgataa ggtgctttct gcttacaata agcacaggga taagcccatc
     8041 agggagcagg cagaaaacat tatccacttg tttactctga ccaacttggg cgcgcctgca
     8101 gccttcaagt acttcgacac caccatagac agaaagcggt acacctctac aaaggaggtc
     8161 ctggacgcca cactgattca tcagtcaatt acggggctct atgaaacaag aatcgacctc
     8221 tctcagctcg gtggagacag cagggctgac cccaagaaga agaggaaggt ggctagccgc
     8281 gccgacgcgc tggacgattt cgatctcgac atgctgggtt ctgatgccct cgatgacttt
     8341 gacctggata tgttgggaag cgacgcattg gatgactttg atctggacat gctcggctcc
     8401 gatgctctgg acgatttcga tctcgatatg ttaatcgcta gcgagggcag aggaagtctt
     8461 ctaacatgcg gtgacgtgga ggagaatccc ggccctggta ccatggccaa gcctttgtct
     8521 caagaagaat ccaccctcat tgaaagagca acggctacaa tcaacagcat ccccatctct
     8581 gaagactaca gcgtcgccag cgcagctctc tctagcgacg gccgcatctt cactggtgtc
     8641 aatgtatatc attttactgg gggaccttgt gcagaactcg tggtgctggg cactgctgct
     8701 gctgcggcag ctggcaacct gacttgtatc gtcgcgatcg gaaatgagaa caggggcatc
     8761 ttgagcccct gcggacggtg ccgacaggtg cttctcgatc tgcatcctgg gatcaaagcc
     8821 atagtgaagg acagtgatgg acagccgacg gcagttggga ttcgtgaatt gctgccctct
     8881 ggttatgtgt gggagggcct gcagctgcag tagttgataa tagataactt cgtatagcat
     8941 acattatacg aagttatgaa ttcgatatca agcttatcga taatcaacct ctggattaca
     9001 aaatttgtga aagattgact ggtattctta actatgttgc tccttttacg ctatgtggat
     9061 acgctgcttt aatgcctttg tatcatgcta ttgcttcccg tatggctttc attttctcct
     9121 ccttgtataa atcctggttg ctgtctcttt atgaggagtt gtggcccgtt gtcaggcaac
     9181 gtggcgtggt gtgcactgtg tttgctgacg caacccccac tggttggggc attgccacca
     9241 cctgtcagct cctttccggg actttcgctt tccccctccc tattgccacg gcggaactca
     9301 tcgccgcctg ccttgcccgc tgctggacag gggctcggct gttgggcact gacaattccg
     9361 tggtgttgtc ggggaaatca tcgtcctttc cttggctgct cgcctgtgtt gccacctgga
     9421 ttctgcgcgg gacgtccttc tgctacgtcc cttcggccct caatccagcg gaccttcctt
     9481 cccgcggcct gctgccggct ctgcggcctc ttccgcgtct tcgccttcgc cctcagacga
     9541 gtcggatctc cctttgggcc gcctccccgc atcgataccg tcgacctcga gacctagaaa
     9601 aacatggagc aatcacaagt agcaatacag cagctaccaa tgctgattgt gcctggctag
     9661 aagcacaaga ggaggaggag gtgggttttc cagtcacacc tcaggtacct ttaagaccaa
     9721 tgacttacaa ggcagctgta gatcttagcc actttttaaa agaaaagggg ggactggaag
     9781 ggctaattca ctcccaacga agacaagata tccttgatct gtggatctac cacacacaag
     9841 gctacttccc tgattggcag aactacacac cagggccagg gatcagatat ccactgacct
     9901 ttggatggtg ctacaagcta gtaccagttg agcaagagaa ggtagaagaa gccaatgaag
     9961 gagagaacac ccgcttgtta caccctgtga gcctgcatgg gatggatgac ccggagagag
    10021 aagtattaga gtggaggttt gacagccgcc tagcatttca tcacatggcc cgagagctgc
    10081 atccggactg tactgggtct ctctggttag accagatctg agcctgggag ctctctggct
    10141 aactagggaa cccactgctt aagcctcaat aaagcttgcc ttgagtgctt caagtagtgt
    10201 gtgcccgtct gttgtgtgac tctggtaact agagatccct cagacccttt tagtcagtgt
    10261 ggaaaatctc tagcagggcc cgtttaaacc cgctgatcag cctcgactgt gccttctagt
    10321 tgccagccat ctgttgtttg cccctccccc gtgccttcct tgaccctgga aggtgccact
    10381 cccactgtcc tttcctaata aaatgaggaa attgcatcgc attgtctgag taggtgtcat
    10441 tctattctgg ggggtggggt ggggcaggac agcaaggggg aggattggga agacaatagc
    10501 aggcatgctg gggatgcggt gggctctatg gcttctgagg cggaaagaac cagctggggc
    10561 tctagggggt atccccacgc gccctgtagc ggcgcattaa gcgcggcggg tgtggtggtt
    10621 acgcgcagcg tgaccgctac acttgccagc gccctagcgc ccgctccttt cgctttcttc
    10681 ccttcctttc tcgccacgtt cgccggcttt ccccgtcaag ctctaaatcg ggggctccct
    10741 ttagggttcc gatttagtgc tttacggcac ctcgacccca aaaaacttga ttagggtgat
    10801 ggttcacgta gtgggccatc gccctgatag acggtttttc gccctttgac gttggagtcc
    10861 acgttcttta atagtggact cttgttccaa actggaacaa cactcaaccc tatctcggtc
    10921 tattcttttg atttataagg gattttgccg atttcggcct attggttaaa aaatgagctg
    10981 atttaacaaa aatttaacgc gaattaattc tgtggaatgt gtgtcagtta gggtgtggaa
    11041 agtccccagg ctccccagca ggcagaagta tgcaaagcat gcatctcaat tagtcagcaa
    11101 ccaggtgtgg aaagtcccca ggctccccag caggcagaag tatgcaaagc atgcatctca
    11161 attagtcagc aaccatagtc ccgcccctaa ctccgcccat cccgccccta actccgccca
    11221 gttccgccca ttctccgccc catggctgac taattttttt tatttatgca gaggccgagg
    11281 ccgcctctgc ctctgagcta ttccagaagt agtgaggagg cttttttgga ggcctaggct
    11341 tttgcaaaaa gctcccggga gcttgtatat ccattttcgg atctgatcag cacgtgttga
    11401 caattaatca tcggcatagt atatcggcat agtataatac gacaaggtga ggaactaaac
    11461 catggccaag ttgaccagtg ccgttccggt gctcaccgcg cgcgacgtcg ccggagcggt
    11521 cgagttctgg accgaccggc tcgggttctc ccgggacttc gtggaggacg acttcgccgg
    11581 tgtggtccgg gacgacgtga ccctgttcat cagcgcggtc caggaccagg tggtgccgga
    11641 caacaccctg gcctgggtgt gggtgcgcgg cctggacgag ctgtacgccg agtggtcgga
    11701 ggtcgtgtcc acgaacttcc gggacgcctc cgggccggcc atgaccgaga tcggcgagca
    11761 gccgtggggg cgggagttcg ccctgcgcga cccggccggc aactgcgtgc acttcgtggc
    11821 cgaggagcag gactgacacg tgctacgaga tttcgattcc accgccgcct tctatgaaag
    11881 gttgggcttc ggaatcgttt tccgggacgc cggctggatg atcctccagc gcggggatct
    11941 catgctggag ttcttcgccc accccaactt gtttattgca gcttataatg gttacaaata
    12001 aagcaatagc atcacaaatt tcacaaataa agcatttttt tcactgcatt ctagttgtgg
    12061 tttgtccaaa ctcatcaatg tatcttatca tgtctgtata ccgtcgacct ctagctagag
    12121 cttggcgtaa tcatggtcat agctgtttcc tgtgtgaaat tgttatccgc tcacaattcc
    12181 acacaacata cgagccggaa gcataaagtg taaagcctgg ggtgcctaat gagtgagcta
    12241 actcacatta attgcgttgc gctcactgcc cgctttccag tcgggaaacc tgtcgtgcca
    12301 gctgcattaa tgaatcggcc aacgcgcggg gagaggcggt ttgcgtattg ggcgctcttc
    12361 cgcttcctcg ctcactgact cgctgcgctc ggtcgttcgg ctgcggcgag cggtatcagc
    12421 tcactcaaag gcggtaatac ggttatccac agaatcaggg gataacgcag gaaagaacat
    12481 gtgagcaaaa ggccagcaaa aggccaggaa ccgtaaaaag gccgcgttgc tggcgttttt
    12541 ccataggctc cgcccccctg acgagcatca caaaaatcga cgctcaagtc agaggtggcg
    12601 aaacccgaca ggactataaa gataccaggc gtttccccct ggaagctccc tcgtgcgctc
    12661 tcctgttccg accctgccgc ttaccggata cctgtccgcc tttctccctt cgggaagcgt
    12721 ggcgctttct catagctcac gctgtaggta tctcagttcg gtgtaggtcg ttcgctccaa
    12781 gctgggctgt gtgcacgaac cccccgttca gcccgaccgc tgcgccttat ccggtaacta
    12841 tcgtcttgag tccaacccgg taagacacga cttatcgcca ctggcagcag ccactggtaa
    12901 caggattagc agagcgaggt atgtaggcgg tgctacagag ttcttgaagt ggtggcctaa
    12961 ctacggctac actagaagaa cagtatttgg tatctgcgct ctgctgaagc cagttacctt
    13021 cggaaaaaga gttggtagct cttgatccgg caaacaaacc accgctggta gcggtggttt
    13081 ttttgtttgc aagcagcaga ttacgcgcag aaaaaaagga tctcaagaag atcctttgat
    13141 cttttctacg gggtctgacg ctcagtggaa cgaaaactca cgttaaggga ttttggtcat
    13201 gagattatca aaaaggatct tcacctagat ccttttaaat taaaaatgaa gttttaaatc
    13261 aatctaaagt atatatgagt aaacttggtc tgacagttac caatgcttaa tcagtgaggc
    13321 acctatctca gcgatctgtc tatttcgttc atccatagtt gcctgactcc ccgtcgtgta
    13381 gataactacg atacgggagg gcttaccatc tggccccagt gctgcaatga taccgcgaga
    13441 cccacgctca ccggctccag atttatcagc aataaaccag ccagccggaa gggccgagcg
    13501 cagaagtggt cctgcaactt tatccgcctc catccagtct attaattgtt gccgggaagc
    13561 tagagtaagt agttcgccag ttaatagttt gcgcaacgtt gttgccattg ctacaggcat
    13621 cgtggtgtca cgctcgtcgt ttggtatggc ttcattcagc tccggttccc aacgatcaag
    13681 gcgagttaca tgatccccca tgttgtgcaa aaaagcggtt agctccttcg gtcctccgat
    13741 cgttgtcaga agtaagttgg ccgcagtgtt atcactcatg gttatggcag cactgcataa
    13801 ttctcttact gtcatgccat ccgtaagatg cttttctgtg actggtgagt actcaaccaa
    13861 gtcattctga gaatagtgta tgcggcgacc gagttgctct tgcccggcgt caatacggga
    13921 taataccgcg ccacatagca gaactttaaa agtgctcatc attggaaaac gttcttcggg
    13981 gcgaaaactc tcaaggatct taccgctgtt gagatccagt tcgatgtaac ccactcgtgc
    14041 acccaactga tcttcagcat cttttacttt caccagcgtt tctgggtgag caaaaacagg
    14101 aaggcaaaat gccgcaaaaa agggaataag ggcgacacgg aaatgttgaa tactcatact
    14161 cttccttttt caatattatt gaagcattta tcagggttat tgtctcatga gcggatacat
    14221 atttgaatgt atttagaaaa ataaacaaat aggggttccg cgcacatttc cccgaaaagt
    14281 gccacctgac
//
