LOCUS       sequence-213540-       14873 bp    DNA     circular SYN 15-DEC-2025
DEFINITION  Lentiviral plasmid for expression of gRNA and dCas9 in mammalian 
            cells; derivative of lentiCRISPR v2.
ACCESSION   .
VERSION     .
KEYWORDS    .
SOURCE      synthetic DNA construct
  ORGANISM  synthetic DNA construct
REFERENCE   1  (bases 1 to 14873)
  AUTHORS   Babaei M, Liu Y, Wuerzberger-Davis SM, McCaslin EZ, DiRusso CJ, Yeo 
            AT, Kagermazova L, Miyamoto S, Gilmore TD
  TITLE     CRISPR/Cas9-based editing of a sensitive transcriptional regulatory 
            element to achieve cell type-specific knockdown of the NEMO scaffold
            protein.
  JOURNAL   PLoS One. 2019 Sep 25;14(9):e0222588. doi: 
            10.1371/journal.pone.0222588. eCollection 2019.
  PUBMED    31553754
REFERENCE   2  (bases 1 to 14873)
  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: "PLoS One.";
            date: "2019-09-25"; volume: "14(9):e0222588. doi"; pages: " 
            10.1371/journal.pone.0222588. eCollection 2019"
COMMENT     Sequence Label: lentiCRISPR v2-dCas9
FEATURES             Location/Qualifiers
     source          1..14873
                     /mol_type="other DNA"
                     /organism="synthetic DNA construct"
     misc_feature    316..433
                     /label=cPPT/CTS
                     /note="central polypurine tract and central termination 
                     sequence of HIV-1"
     promoter        484..732
                     /label=U6 promoter
                     /note="RNA polymerase III promoter for human U6 snRNA 
                     (Domitrovich & Kunkel, 2003)"
     primer_bind     484..504
                     /label=hU6-F
                     /note="Human U6 promoter, forward primer"
     primer_bind     655..674
                     /label=LKO.1 5'
                     /note="Human U6 promoter, forward primer"
     misc_RNA        2614..2689
                     /label=gRNA scaffold
                     /note="guide RNA scaffold for the Streptococcus pyogenes 
                     CRISPR/Cas9 system"
     promoter        2751..2962
                     /label=EF-1-alpha core promoter
                     /note="core promoter for human elongation factor 
                     EF-1-alpha"
     regulatory      2981..2990
                     /label=Kozak sequence
                     /note="vertebrate consensus sequence for strong initiation 
                     of translation (Kozak, 1987)"
                     /regulatory_class="other"
     CDS             2987..7090
                     /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="MDKKYSIGLGIGTNSVGWAVITDEYKVPSKKFKVLGNTDRHSIKK
                     NLIGALLFDSGETAEATRLKRTARRRYTRRKNRICYLQEIFSNEMAKVDDSFFHRLEES
                     FLVEEDKKHERHPIFGNIVDEVAYHEKYPTIYHLRKKLVDSTDKADLRLIYLALAHMIK
                     FRGHFLIEGDLNPDNSDVDKLFIQLVQTYNQLFEENPINASGVDAKAILSARLSKSRRL
                     ENLIAQLPGEKKNGLFGNLIALSLGLTPNFKSNFDLAEDAKLQLSKDTYDDDLDNLLAQ
                     IGDQYADLFLAAKNLSDAILLSDILRVNTEITKAPLSASMIKRYDEHHQDLTLLKALVR
                     QQLPEKYKEIFFDQSKNGYAGYIDGGASQEEFYKFIKPILEKMDGTEELLVKLNREDLL
                     RKQRTFDNGSIPHQIHLGELHAILRRQEDFYPFLKDNREKIEKILTFRIPYYVGPLARG
                     NSRFAWMTRKSEETITPWNFEEVVDKGASAQSFIERMTNFDKNLPNEKVLPKHSLLYEY
                     FTVYNELTKVKYVTEGMRKPAFLSGEQKKAIVDLLFKTNRKVTVKQLKEDYFKKIECFD
                     SVEISGVEDRFNASLGTYHDLLKIIKDKDFLDNEENEDILEDIVLTLTLFEDREMIEER
                     LKTYAHLFDDKVMKQLKRRRYTGWGRLSRKLINGIRDKQSGKTILDFLKSDGFANRNFM
                     QLIHDDSLTFKEDIQKAQVSGQGDSLHEHIANLAGSPAIKKGILQTVKVVDELVKVMGR
                     HKPENIVIEMARENQTTQKGQKNSRERMKRIEEGIKELGSQILKEHPVENTQLQNEKLY
                     LYYLQNGRDMYVDQELDINRLSDYDVDAIVPQSFLKDDSIDNKVLTRSDKNRGKSDNVP
                     SEEVVKKMKNYWRQLLNAKLITQRKFDNLTKAERGGLSELDKAGFIKRQLVETRQITKH
                     VAQILDSRMNTKYDENDKLIREVKVITLKSKLVSDFRKDFQFYKVREINNYHHAHDAYL
                     NAVVGTALIKKYPKLESEFVYGDYKVYDVRKMIAKSEQEIGKATAKYFFYSNIMNFFKT
                     EITLANGEIRKRPLIETNGETGEIVWDKGRDFATVRKVLSMPQVNIVKKTEVQTGGFSK
                     ESILPKRNSDKLIARKKDWDPKKYGGFDSPTVAYSVLVVAKVEKGKSKKLKSVKELLGI
                     TIMERSSFEKNPIDFLEAKGYKEVKKDLIIKLPKYSLFELENGRKRMLASAGELQKGNE
                     LALPSKYVNFLYLASHYEKLKGSPEDNEQKQLFVEQHKHYLDEIIEQISEFSKRVILAD
                     ANLDKVLSAYNKHRDKPIREQAENIIHLFTLTNLGAPAAFKYFDTTIDRKRYTSTKEVL
                     DATLIHQSITGLYETRIDLSQLGGD"
     CDS             7091..7138
                     /codon_start=1
                     /product="bipartite nuclear localization signal from 
                     nucleoplasmin"
                     /label=nucleoplasmin NLS
                     /translation="KRPAATKKAGQAKKKK"
     CDS             7139..7162
                     /codon_start=1
                     /product="FLAG(R) epitope tag, followed by an enterokinase 
                     cleavage site"
                     /label=FLAG
                     /translation="DYKDDDDK"
     CDS             7172..7228
                     /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             7229..7825
                     /codon_start=1
                     /gene="pac from Streptomyces alboniger"
                     /product="puromycin N-acetyltransferase"
                     /label=PuroR
                     /note="confers resistance to puromycin"
                     /translation="TEYKPTVRLATRDDVPRAVRTLAAAFADYPATRHTVDPDRHIERV
                     TELQELFLTRVGLDIGKVWVADDGAAVAVWTTPESVEAGAVFAEIGPRMAELSGSRLAA
                     QQQMEGLLAPHRPKEPAWFLATVGVSPDHQGKGLGSAVVLPGVEAAERAGVPAFLETSA
                     PRNLPFYERLGFTVTADVEVPEGPRTWCMTRKPGA"
     primer_bind     7722..7742
                     /label=Puro-F
                     /note="Puromycin resistance gene, forward primer"
     misc_feature    7841..8429
                     /label=WPRE
                     /note="woodchuck hepatitis virus posttranscriptional 
                     regulatory element"
     primer_bind     complement(7894..7914)
                     /label=WPRE-R
                     /note="WPRE, reverse primer"
     CDS             complement(8312..8323)
                     /codon_start=1
                     /product="Factor Xa recognition and cleavage site"
                     /label=Factor Xa site
                     /translation="IEGR"
     LTR             8501..8734
                     /label=3' LTR (Delta-U3)
                     /note="self-inactivating 3' long terminal repeat (LTR) from
                     HIV-1"
     primer_bind     complement(8760..8777)
                     /label=BGH-rev
                     /note="Bovine growth hormone terminator, reverse primer. 
                     Also called BGH reverse"
     polyA_signal    8766..8990
                     /label=bGH poly(A) signal
                     /note="bovine growth hormone polyadenylation signal"
     rep_origin      9036..9464
                     /direction=RIGHT
                     /label=f1 ori
                     /note="f1 bacteriophage origin of replication; arrow 
                     indicates direction of (+) strand synthesis"
     primer_bind     complement(9123..9142)
                     /label=F1ori-R
                     /note="F1 origin, reverse primer"
     primer_bind     9333..9354
                     /label=F1ori-F
                     /note="F1 origin, forward primer"
     primer_bind     complement(9473..9493)
                     /label=pBABE 3'
                     /note="SV40 enhancer, reverse primer for pBABE vectors"
     promoter        9478..9807
                     /label=SV40 promoter
                     /note="SV40 enhancer and early promoter"
     rep_origin      9658..9793
                     /label=SV40 ori
                     /note="SV40 origin of replication"
     primer_bind     9720..9739
                     /label=SV40pro-F
                     /note="SV40 promoter/origin, forward primer"
     promoter        9855..9902
                     /label=EM7 promoter
                     /note="synthetic bacterial promoter "
     CDS             9921..10295
                     /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    10425..10546
                     /label=SV40 poly(A) signal
                     /note="SV40 polyadenylation signal"
     primer_bind     complement(10462..10481)
                     /label=SV40pA-R
                     /note="SV40 polyA, reverse primer"
     primer_bind     10516..10535
                     /label=EBV-rev
                     /note="SV40 polyA terminator, reverse primer"
     primer_bind     complement(10595..10611)
                     /label=M13 rev
                     /note="common sequencing primer, one of multiple similar 
                     variants"
     primer_bind     complement(10595..10611)
                     /label=M13 Reverse
                     /note="In lacZ gene. Also called M13-rev"
     primer_bind     complement(10608..10630)
                     /label=M13/pUC Reverse
                     /note="In lacZ gene"
     protein_bind    10619..10635
                     /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(10643..10649,10650..10667,10668..10673))
                     /label=lac promoter
                     /note="promoter for the E. coli lac operon"
     protein_bind    10688..10709
                     /label=CAP binding site
                     /bound_moiety="E. coli catabolite activator protein"
                     /note="CAP binding activates transcription in the presence 
                     of cAMP."
     primer_bind     complement(10826..10843)
                     /label=L4440
                     /note="L4440 vector, forward primer"
     rep_origin      complement(10997..11585)
                     /direction=LEFT
                     /label=ori
                     /note="high-copy-number ColE1/pMB1/pBR322/pUC origin of 
                     replication"
     primer_bind     complement(11077..11096)
                     /label=pBR322ori-F
                     /note="pBR322 origin, forward primer"
     CDS             complement(join(11756..12547,12548..12616))
                     /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     12379..12398
                     /label=Amp-R
                     /note="Ampicillin resistance gene, reverse primer"
     promoter        complement(12617..12721)
                     /gene="bla"
                     /label=AmpR promoter
     primer_bind     complement(12796..12815)
                     /label=pRS-marker
                     /note="pRS vectors, use to sequence yeast selectable 
                     marker"
     enhancer        12987..13366
                     /label=CMV enhancer
                     /note="human cytomegalovirus immediate early enhancer"
     promoter        13368..13566
                     /label=CMV promoter
                     /note="human cytomegalovirus (CMV) immediate early 
                     promoter"
     primer_bind     13521..13541
                     /label=CMV-F
                     /note="Human CMV immediate early promoter, forward primer"
     LTR             13584..13764
                     /label=5' LTR (truncated)
                     /note="truncated 5' long terminal repeat (LTR) from HIV-1"
     misc_feature    13811..13936
                     /label=HIV-1 Psi
                     /note="packaging signal of human immunodeficiency virus 
                     type 1"
     misc_feature    14429..14662
                     /label=RRE
                     /note="The Rev response element (RRE) of HIV-1 allows for 
                     Rev-dependent mRNA export from the nucleus to the 
                     cytoplasm."
     CDS             join(14847..14873,1..18)
                     /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"
ORIGIN
        1 gataaatggg caagtttgtg gaattggttt aacataacaa attggctgtg gtatataaaa
       61 ttattcataa tgatagtagg aggcttggta ggtttaagaa tagtttttgc tgtactttct
      121 atagtgaata gagttaggca gggatattca ccattatcgt ttcagaccca cctcccaacc
      181 ccgaggggac ccgacaggcc cgaaggaata gaagaagaag gtggagagag agacagagac
      241 agatccattc gattagtgaa cggatcggca ctgcgtgcgc caattctgca gacaaatggc
      301 agtattcatc cacaatttta aaagaaaagg ggggattggg gggtacagtg caggggaaag
      361 aatagtagac ataatagcaa cagacataca aactaaagaa ttacaaaaac aaattacaaa
      421 aattcaaaat tttcgggttt attacaggga cagcagagat ccagtttggt taattaaggt
      481 accgagggcc tatttcccat gattccttca tatttgcata tacgatacaa ggctgttaga
      541 gagataatta gaattaattt gactgtaaac acaaagatat tagtacaaaa tacgtgacgt
      601 agaaagtaat aatttcttgg gtagtttgca gttttaaaat tatgttttaa aatggactat
      661 catatgctta ccgtaacttg aaagtatttc gatttcttgg ctttatatat cttgtggaaa
      721 ggacgaaaca ccggagacgg ttgtaaatga gcacacaaaa tacacatgct aaaatattat
      781 attctatgac ctttataaaa tcaaccaaaa tcttcttttt aataacttta gtatcaataa
      841 ttagaatttt tatgttcctt tttgcaaact tttaataaaa atgagcaaaa taaaaaaacg
      901 ctagttttag taactcgcgt tgttttcttc acctttaata atagctactc caccacttgt
      961 tcctaagcgg tcagctcctg cttcaatcat tttttgagca tcttcaaatg ttctaactcc
     1021 accagctgct ttaactaaag cattgtcttt aacaactgac ttcattagtt taacatcttc
     1081 aaatgttgca cctgattttg aaaatcctgt tgatgtttta acaaattcta atccagcttc
     1141 aacagctatt tcacaagctt tcatgatttc ttcttttgtt aataaacaat tttccataat
     1201 acatttaaca acatgtgatc cagctgcttt ttttacagct ttcatgtctt ctaaaactaa
     1261 ttcataattt ttgtctttta atgcaccaat atttaatacc atatcaattt ctgttgcacc
     1321 atctttaatt gcttcagaaa cttcgaatgc ttttgtagct gttgtgcatg cacctagagg
     1381 aaaacctaca acatttgtta ttcctacatt tgtgcctttt aataattctt tacaatagct
     1441 tgttcaatat gaattaacac aaactgttgc aaaatcaaat tcaattgctt catcacataa
     1501 ttgtttaatt tcagctttcg tagcatcttg ttttaataat gtgtgatcta tatatttgtt
     1561 tagtttcatt ttttctccta tatattcatt tttaatttta attctttaat aatttcgtct
     1621 actttaactt tagcgttttg aacagattca ccaacaccta taaaataaat ttttagttta
     1681 ggttcagttc cacttgggcg aacagcaaat catgacttat cttctaaata aaattttagt
     1741 aagtcttgtc ctggcatatt atacattcca tcgatgtagt cttcaacatt aacaacttta
     1801 agtccagcaa tttgagttaa gggtgttgct ctcaatgatt tcattaatgg ttcaattttt
     1861 aatttctttt cttctggttt aaaattcaag tttaaagtga aagtgtaata tgcacccatt
     1921 tctttaaata aatcttctaa atagtctact aatgttttat tttgtttttt ataaaatcaa
     1981 gcagcctctg ctattaatat agaagcttgt attccatctt tatctctagc tgagtcatca
     2041 attacatatc cataactttc ttcataagca aaaacaaaat ttaatccgtt atcttcttct
     2101 ttagcaattt ctctacccat tcatttaaat ccagttaaag tttttacaat attaactcca
     2161 tatttttcat gagcgattct atcacccaaa tcacttgtta caaaacttga atatagagcc
     2221 ggattttttg gaatgctatt taagcgtttt agatttgata attttcaatc aattaaaatt
     2281 ggtcctgttt gatttccatc taatcttaca aaatgaccat catgttttat tgccattcca
     2341 aatctgtcag catctgggtc attcataata ataatatctg catcatgttt aataccatat
     2401 tcaagcggta tttttcatgc aggatcaaat tctggatttg gatttacaac atttttaaat
     2461 gtttcatctt caaatgcatg ctcttcaacc tcaataacgt tatatcctga ttcacgtaat
     2521 atttttgggg taaatttagt tcctgttcca ttaactgcgc taaaaataat ttttaaatct
     2581 tttttagctt cttgctcttt tttgtacgtc tctgttttag agctagaaat agcaagttaa
     2641 aataaggcta gtccgttatc aacttgaaaa agtggcaccg agtcggtgct tttttgaatt
     2701 cgctagctag gtcttgaaag gagtgggaat tggctccggt gcccgtcagt gggcagagcg
     2761 cacatcgccc acagtccccg agaagttggg gggaggggtc ggcaattgat ccggtgccta
     2821 gagaaggtgg cgcggggtaa actgggaaag tgatgtcgtg tactggctcc gcctttttcc
     2881 cgagggtggg ggagaaccgt atataagtgc agtagtcgcc gtgaacgttc tttttcgcaa
     2941 cgggtttgcc gccagaacac aggaccggtt ctagagcgct gccaccatgg acaagaagta
     3001 cagcatcggc ctgggcatcg gcaccaactc tgtgggctgg gccgtgatca ccgacgagta
     3061 caaggtgccc agcaagaaat tcaaggtgct gggcaacacc gaccggcaca gcatcaagaa
     3121 gaacctgatc ggagccctgc tgttcgacag cggcgaaaca gccgaggcca cccggctgaa
     3181 gagaaccgcc agaagaagat acaccagacg gaagaaccgg atctgctatc tgcaagagat
     3241 cttcagcaac gagatggcca aggtggacga cagcttcttc cacagactgg aagagtcctt
     3301 cctggtggaa gaggataaga agcacgagcg gcaccccatc ttcggcaaca tcgtggacga
     3361 ggtggcctac cacgagaagt accccaccat ctaccacctg agaaagaaac tggtggacag
     3421 caccgacaag gccgacctgc ggctgatcta tctggccctg gcccacatga tcaagttccg
     3481 gggccacttc ctgatcgagg gcgacctgaa ccccgacaac agcgacgtgg acaagctgtt
     3541 catccagctg gtgcagacct acaaccagct gttcgaggaa aaccccatca acgccagcgg
     3601 cgtggacgcc aaggccatcc tgtctgccag actgagcaag agcagacggc tggaaaatct
     3661 gatcgcccag ctgcccggcg agaagaagaa tggcctgttc ggaaacctga ttgccctgag
     3721 cctgggcctg acccccaact tcaagagcaa cttcgacctg gccgaggatg ccaaactgca
     3781 gctgagcaag gacacctacg acgacgacct ggacaacctg ctggcccaga tcggcgacca
     3841 gtacgccgac ctgtttctgg ccgccaagaa cctgtccgac gccatcctgc tgagcgacat
     3901 cctgagagtg aacaccgaga tcaccaaggc ccccctgagc gcctctatga tcaagagata
     3961 cgacgagcac caccaggacc tgaccctgct gaaagctctc gtgcggcagc agctgcctga
     4021 gaagtacaaa gagattttct tcgaccagag caagaacggc tacgccggct acattgacgg
     4081 cggagccagc caggaagagt tctacaagtt catcaagccc atcctggaaa agatggacgg
     4141 caccgaggaa ctgctcgtga agctgaacag agaggacctg ctgcggaagc agcggacctt
     4201 cgacaacggc agcatccccc accagatcca cctgggagag ctgcacgcca ttctgcggcg
     4261 gcaggaagat ttttacccat tcctgaagga caaccgggaa aagatcgaga agatcctgac
     4321 cttccgcatc ccctactacg tgggccctct ggccagggga aacagcagat tcgcctggat
     4381 gaccagaaag agcgaggaaa ccatcacccc ctggaacttc gaggaagtgg tggacaaggg
     4441 cgcttccgcc cagagcttca tcgagcggat gaccaacttc gataagaacc tgcccaacga
     4501 gaaggtgctg cccaagcaca gcctgctgta cgagtacttc accgtgtata acgagctgac
     4561 caaagtgaaa tacgtgaccg agggaatgag aaagcccgcc ttcctgagcg gcgagcagaa
     4621 aaaggccatc gtggacctgc tgttcaagac caaccggaaa gtgaccgtga agcagctgaa
     4681 agaggactac ttcaagaaaa tcgagtgctt cgactccgtg gaaatctccg gcgtggaaga
     4741 tcggttcaac gcctccctgg gcacatacca cgatctgctg aaaattatca aggacaagga
     4801 cttcctggac aatgaggaaa acgaggacat tctggaagat atcgtgctga ccctgacact
     4861 gtttgaggac agagagatga tcgaggaacg gctgaaaacc tatgcccacc tgttcgacga
     4921 caaagtgatg aagcagctga agcggcggag atacaccggc tggggcaggc tgagccggaa
     4981 gctgatcaac ggcatccggg acaagcagtc cggcaagaca atcctggatt tcctgaagtc
     5041 cgacggcttc gccaacagaa acttcatgca gctgatccac gacgacagcc tgacctttaa
     5101 agaggacatc cagaaagccc aggtgtccgg ccagggcgat agcctgcacg agcacattgc
     5161 caatctggcc ggcagccccg ccattaagaa gggcatcctg cagacagtga aggtggtgga
     5221 cgagctcgtg aaagtgatgg gccggcacaa gcccgagaac atcgtgatcg aaatggccag
     5281 agagaaccag accacccaga agggacagaa gaacagccgc gagagaatga agcggatcga
     5341 agagggcatc aaagagctgg gcagccagat cctgaaagaa caccccgtgg aaaacaccca
     5401 gctgcagaac gagaagctgt acctgtacta cctgcagaat gggcgggata tgtacgtgga
     5461 ccaggaactg gacatcaacc ggctgtccga ctacgatgtg gacgccatcg tgcctcagag
     5521 ctttctgaag gacgactcca tcgacaacaa ggtgctgacc agaagcgaca agaaccgggg
     5581 caagagcgac aacgtgccct ccgaagaggt cgtgaagaag atgaagaact actggcggca
     5641 gctgctgaac gccaagctga ttacccagag aaagttcgac aatctgacca aggccgagag
     5701 aggcggcctg agcgaactgg ataaggccgg cttcatcaag agacagctgg tggaaacccg
     5761 gcagatcaca aagcacgtgg cacagatcct ggactcccgg atgaacacta agtacgacga
     5821 gaatgacaag ctgatccggg aagtgaaagt gatcaccctg aagtccaagc tggtgtccga
     5881 tttccggaag gatttccagt tttacaaagt gcgcgagatc aacaactacc accacgccca
     5941 cgacgcctac ctgaacgccg tcgtgggaac cgccctgatc aaaaagtacc ctaagctgga
     6001 aagcgagttc gtgtacggcg actacaaggt gtacgacgtg cggaagatga tcgccaagag
     6061 cgagcaggaa atcggcaagg ctaccgccaa gtacttcttc tacagcaaca tcatgaactt
     6121 tttcaagacc gagattaccc tggccaacgg cgagatccgg aagcggcctc tgatcgagac
     6181 aaacggcgaa accggggaga tcgtgtggga taagggccgg gattttgcca ccgtgcggaa
     6241 agtgctgagc atgccccaag tgaatatcgt gaaaaagacc gaggtgcaga caggcggctt
     6301 cagcaaagag tctatcctgc ccaagaggaa cagcgataag ctgatcgcca gaaagaagga
     6361 ctgggaccct aagaagtacg gcggcttcga cagccccacc gtggcctatt ctgtgctggt
     6421 ggtggccaaa gtggaaaagg gcaagtccaa gaaactgaag agtgtgaaag agctgctggg
     6481 gatcaccatc atggaaagaa gcagcttcga gaagaatccc atcgactttc tggaagccaa
     6541 gggctacaaa gaagtgaaaa aggacctgat catcaagctg cctaagtact ccctgttcga
     6601 gctggaaaac ggccggaaga gaatgctggc ctctgccggc gaactgcaga agggaaacga
     6661 actggccctg ccctccaaat atgtgaactt cctgtacctg gccagccact atgagaagct
     6721 gaagggctcc cccgaggata atgagcagaa acagctgttt gtggaacagc acaagcacta
     6781 cctggacgag atcatcgagc agatcagcga gttctccaag agagtgatcc tggccgacgc
     6841 taatctggac aaagtgctgt ccgcctacaa caagcaccgg gataagccca tcagagagca
     6901 ggccgagaat atcatccacc tgtttaccct gaccaatctg ggagcccctg ccgccttcaa
     6961 gtactttgac accaccatcg accggaagag gtacaccagc accaaagagg tgctggacgc
     7021 caccctgatc caccagagca tcaccggcct gtacgagaca cggatcgacc tgtctcagct
     7081 gggaggcgac aagcgacctg ccgccacaaa gaaggctgga caggctaaga agaagaaaga
     7141 ttacaaagac gatgacgata agggatccgg cgcaacaaac ttctctctgc tgaaacaagc
     7201 cggagatgtc gaagagaatc ctggaccgac cgagtacaag cccacggtgc gcctcgccac
     7261 ccgcgacgac gtccccaggg ccgtacgcac cctcgccgcc gcgttcgccg actaccccgc
     7321 cacgcgccac accgtcgatc cggaccgcca catcgagcgg gtcaccgagc tgcaagaact
     7381 cttcctcacg cgcgtcgggc tcgacatcgg caaggtgtgg gtcgcggacg acggcgccgc
     7441 ggtggcggtc tggaccacgc cggagagcgt cgaagcgggg gcggtgttcg ccgagatcgg
     7501 cccgcgcatg gccgagttga gcggttcccg gctggccgcg cagcaacaga tggaaggcct
     7561 cctggcgccg caccggccca aggagcccgc gtggttcctg gccaccgtcg gagtctcgcc
     7621 cgaccaccag ggcaagggtc tgggcagcgc cgtcgtgctc cccggagtgg aggcggccga
     7681 gcgcgccggg gtgcccgcct tcctggagac ctccgcgccc cgcaacctcc ccttctacga
     7741 gcggctcggc ttcaccgtca ccgccgacgt cgaggtgccc gaaggaccgc gcacctggtg
     7801 catgacccgc aagcccggtg cctgaacgcg ttaagtcgac aatcaacctc tggattacaa
     7861 aatttgtgaa agattgactg gtattcttaa ctatgttgct ccttttacgc tatgtggata
     7921 cgctgcttta atgcctttgt atcatgctat tgcttcccgt atggctttca ttttctcctc
     7981 cttgtataaa tcctggttgc tgtctcttta tgaggagttg tggcccgttg tcaggcaacg
     8041 tggcgtggtg tgcactgtgt ttgctgacgc aacccccact ggttggggca ttgccaccac
     8101 ctgtcagctc ctttccggga ctttcgcttt ccccctccct attgccacgg cggaactcat
     8161 cgccgcctgc cttgcccgct gctggacagg ggctcggctg ttgggcactg acaattccgt
     8221 ggtgttgtcg gggaaatcat cgtcctttcc ttggctgctc gcctgtgttg ccacctggat
     8281 tctgcgcggg acgtccttct gctacgtccc ttcggccctc aatccagcgg accttccttc
     8341 ccgcggcctg ctgccggctc tgcggcctct tccgcgtctt cgccttcgcc ctcagacgag
     8401 tcggatctcc ctttgggccg cctccccgcg tcgactttaa gaccaatgac ttacaaggca
     8461 gctgtagatc ttagccactt tttaaaagaa aaggggggac tggaagggct aattcactcc
     8521 caacgaagac aagatctgct ttttgcttgt actgggtctc tctggttaga ccagatctga
     8581 gcctgggagc tctctggcta actagggaac ccactgctta agcctcaata aagcttgcct
     8641 tgagtgcttc aagtagtgtg tgcccgtctg ttgtgtgact ctggtaacta gagatccctc
     8701 agaccctttt agtcagtgtg gaaaatctct agcagggccc gtttaaaccc gctgatcagc
     8761 ctcgactgtg ccttctagtt gccagccatc tgttgtttgc ccctcccccg tgccttcctt
     8821 gaccctggaa ggtgccactc ccactgtcct ttcctaataa aatgaggaaa ttgcatcgca
     8881 ttgtctgagt aggtgtcatt ctattctggg gggtggggtg gggcaggaca gcaaggggga
     8941 ggattgggaa gacaatagca ggcatgctgg ggatgcggtg ggctctatgg cttctgaggc
     9001 ggaaagaacc agctggggct ctagggggta tccccacgcg ccctgtagcg gcgcattaag
     9061 cgcggcgggt gtggtggtta cgcgcagcgt gaccgctaca cttgccagcg ccctagcgcc
     9121 cgctcctttc gctttcttcc cttcctttct cgccacgttc gccggctttc cccgtcaagc
     9181 tctaaatcgg gggctccctt tagggttccg atttagtgct ttacggcacc tcgaccccaa
     9241 aaaacttgat tagggtgatg gttcacgtag tgggccatcg ccctgataga cggtttttcg
     9301 ccctttgacg ttggagtcca cgttctttaa tagtggactc ttgttccaaa ctggaacaac
     9361 actcaaccct atctcggtct attcttttga tttataaggg attttgccga tttcggccta
     9421 ttggttaaaa aatgagctga tttaacaaaa atttaacgcg aattaattct gtggaatgtg
     9481 tgtcagttag ggtgtggaaa gtccccaggc tccccagcag gcagaagtat gcaaagcatg
     9541 catctcaatt agtcagcaac caggtgtgga aagtccccag gctccccagc aggcagaagt
     9601 atgcaaagca tgcatctcaa ttagtcagca accatagtcc cgcccctaac tccgcccatc
     9661 ccgcccctaa ctccgcccag ttccgcccat tctccgcccc atggctgact aatttttttt
     9721 atttatgcag aggccgaggc cgcctctgcc tctgagctat tccagaagta gtgaggaggc
     9781 ttttttggag gcctaggctt ttgcaaaaag ctcccgggag cttgtatatc cattttcgga
     9841 tctgatcagc acgtgttgac aattaatcat cggcatagta tatcggcata gtataatacg
     9901 acaaggtgag gaactaaacc atggccaagt tgaccagtgc cgttccggtg ctcaccgcgc
     9961 gcgacgtcgc cggagcggtc gagttctgga ccgaccggct cgggttctcc cgggacttcg
    10021 tggaggacga cttcgccggt gtggtccggg acgacgtgac cctgttcatc agcgcggtcc
    10081 aggaccaggt ggtgccggac aacaccctgg cctgggtgtg ggtgcgcggc ctggacgagc
    10141 tgtacgccga gtggtcggag gtcgtgtcca cgaacttccg ggacgcctcc gggccggcca
    10201 tgaccgagat cggcgagcag ccgtgggggc gggagttcgc cctgcgcgac ccggccggca
    10261 actgcgtgca cttcgtggcc gaggagcagg actgacacgt gctacgagat ttcgattcca
    10321 ccgccgcctt ctatgaaagg ttgggcttcg gaatcgtttt ccgggacgcc ggctggatga
    10381 tcctccagcg cggggatctc atgctggagt tcttcgccca ccccaacttg tttattgcag
    10441 cttataatgg ttacaaataa agcaatagca tcacaaattt cacaaataaa gcattttttt
    10501 cactgcattc tagttgtggt ttgtccaaac tcatcaatgt atcttatcat gtctgtatac
    10561 cgtcgacctc tagctagagc ttggcgtaat catggtcata gctgtttcct gtgtgaaatt
    10621 gttatccgct cacaattcca cacaacatac gagccggaag cataaagtgt aaagcctggg
    10681 gtgcctaatg agtgagctaa ctcacattaa ttgcgttgcg ctcactgccc gctttccagt
    10741 cgggaaacct gtcgtgccag ctgcattaat gaatcggcca acgcgcgggg agaggcggtt
    10801 tgcgtattgg gcgctcttcc gcttcctcgc tcactgactc gctgcgctcg gtcgttcggc
    10861 tgcggcgagc ggtatcagct cactcaaagg cggtaatacg gttatccaca gaatcagggg
    10921 ataacgcagg aaagaacatg tgagcaaaag gccagcaaaa ggccaggaac cgtaaaaagg
    10981 ccgcgttgct ggcgtttttc cataggctcc gcccccctga cgagcatcac aaaaatcgac
    11041 gctcaagtca gaggtggcga aacccgacag gactataaag ataccaggcg tttccccctg
    11101 gaagctccct cgtgcgctct cctgttccga ccctgccgct taccggatac ctgtccgcct
    11161 ttctcccttc gggaagcgtg gcgctttctc atagctcacg ctgtaggtat ctcagttcgg
    11221 tgtaggtcgt tcgctccaag ctgggctgtg tgcacgaacc ccccgttcag cccgaccgct
    11281 gcgccttatc cggtaactat cgtcttgagt ccaacccggt aagacacgac ttatcgccac
    11341 tggcagcagc cactggtaac aggattagca gagcgaggta tgtaggcggt gctacagagt
    11401 tcttgaagtg gtggcctaac tacggctaca ctagaagaac agtatttggt atctgcgctc
    11461 tgctgaagcc agttaccttc ggaaaaagag ttggtagctc ttgatccggc aaacaaacca
    11521 ccgctggtag cggtggtttt tttgtttgca agcagcagat tacgcgcaga aaaaaaggat
    11581 ctcaagaaga tcctttgatc ttttctacgg ggtctgacgc tcagtggaac gaaaactcac
    11641 gttaagggat tttggtcatg agattatcaa aaaggatctt cacctagatc cttttaaatt
    11701 aaaaatgaag ttttaaatca atctaaagta tatatgagta aacttggtct gacagttacc
    11761 aatgcttaat cagtgaggca cctatctcag cgatctgtct atttcgttca tccatagttg
    11821 cctgactccc cgtcgtgtag ataactacga tacgggaggg cttaccatct ggccccagtg
    11881 ctgcaatgat accgcgagac ccacgctcac cggctccaga tttatcagca ataaaccagc
    11941 cagccggaag ggccgagcgc agaagtggtc ctgcaacttt atccgcctcc atccagtcta
    12001 ttaattgttg ccgggaagct agagtaagta gttcgccagt taatagtttg cgcaacgttg
    12061 ttgccattgc tacaggcatc gtggtgtcac gctcgtcgtt tggtatggct tcattcagct
    12121 ccggttccca acgatcaagg cgagttacat gatcccccat gttgtgcaaa aaagcggtta
    12181 gctccttcgg tcctccgatc gttgtcagaa gtaagttggc cgcagtgtta tcactcatgg
    12241 ttatggcagc actgcataat tctcttactg tcatgccatc cgtaagatgc ttttctgtga
    12301 ctggtgagta ctcaaccaag tcattctgag aatagtgtat gcggcgaccg agttgctctt
    12361 gcccggcgtc aatacgggat aataccgcgc cacatagcag aactttaaaa gtgctcatca
    12421 ttggaaaacg ttcttcgggg cgaaaactct caaggatctt accgctgttg agatccagtt
    12481 cgatgtaacc cactcgtgca cccaactgat cttcagcatc ttttactttc accagcgttt
    12541 ctgggtgagc aaaaacagga aggcaaaatg ccgcaaaaaa gggaataagg gcgacacgga
    12601 aatgttgaat actcatactc ttcctttttc aatattattg aagcatttat cagggttatt
    12661 gtctcatgag cggatacata tttgaatgta tttagaaaaa taaacaaata ggggttccgc
    12721 gcacatttcc ccgaaaagtg ccacctgacg tcgacggatc gggagatctc ccgatcccct
    12781 atggtgcact ctcagtacaa tctgctctga tgccgcatag ttaagccagt atctgctccc
    12841 tgcttgtgtg ttggaggtcg ctgagtagtg cgcgagcaaa atttaagcta caacaaggca
    12901 aggcttgacc gacaattgca tgaagaatct gcttagggtt aggcgttttg cgctgcttcg
    12961 cgatgtacgg gccagatata cgcgttgaca ttgattattg actagttatt aatagtaatc
    13021 aattacgggg tcattagttc atagcccata tatggagttc cgcgttacat aacttacggt
    13081 aaatggcccg cctggctgac cgcccaacga cccccgccca ttgacgtcaa taatgacgta
    13141 tgttcccata gtaacgccaa tagggacttt ccattgacgt caatgggtgg agtatttacg
    13201 gtaaactgcc cacttggcag tacatcaagt gtatcatatg ccaagtacgc cccctattga
    13261 cgtcaatgac ggtaaatggc ccgcctggca ttatgcccag tacatgacct tatgggactt
    13321 tcctacttgg cagtacatct acgtattagt catcgctatt accatggtga tgcggttttg
    13381 gcagtacatc aatgggcgtg gatagcggtt tgactcacgg ggatttccaa gtctccaccc
    13441 cattgacgtc aatgggagtt tgttttggca ccaaaatcaa cgggactttc caaaatgtcg
    13501 taacaactcc gccccattga cgcaaatggg cggtaggcgt gtacggtggg aggtctatat
    13561 aagcagcgcg ttttgcctgt actgggtctc tctggttaga ccagatctga gcctgggagc
    13621 tctctggcta actagggaac ccactgctta agcctcaata aagcttgcct tgagtgcttc
    13681 aagtagtgtg tgcccgtctg ttgtgtgact ctggtaacta gagatccctc agaccctttt
    13741 agtcagtgtg gaaaatctct agcagtggcg cccgaacagg gacttgaaag cgaaagggaa
    13801 accagaggag ctctctcgac gcaggactcg gcttgctgaa gcgcgcacgg caagaggcga
    13861 ggggcggcga ctggtgagta cgccaaaaat tttgactagc ggaggctaga aggagagaga
    13921 tgggtgcgag agcgtcagta ttaagcgggg gagaattaga tcgcgatggg aaaaaattcg
    13981 gttaaggcca gggggaaaga aaaaatataa attaaaacat atagtatggg caagcaggga
    14041 gctagaacga ttcgcagtta atcctggcct gttagaaaca tcagaaggct gtagacaaat
    14101 actgggacag ctacaaccat cccttcagac aggatcagaa gaacttagat cattatataa
    14161 tacagtagca accctctatt gtgtgcatca aaggatagag ataaaagaca ccaaggaagc
    14221 tttagacaag atagaggaag agcaaaacaa aagtaagacc accgcacagc aagcggccgc
    14281 tgatcttcag acctggagga ggagatatga gggacaattg gagaagtgaa ttatataaat
    14341 ataaagtagt aaaaattgaa ccattaggag tagcacccac caaggcaaag agaagagtgg
    14401 tgcagagaga aaaaagagca gtgggaatag gagctttgtt ccttgggttc ttgggagcag
    14461 caggaagcac tatgggcgca gcgtcaatga cgctgacggt acaggccaga caattattgt
    14521 ctggtatagt gcagcagcag aacaatttgc tgagggctat tgaggcgcaa cagcatctgt
    14581 tgcaactcac agtctggggc atcaagcagc tccaggcaag aatcctggct gtggaaagat
    14641 acctaaagga tcaacagctc ctggggattt ggggttgctc tggaaaactc atttgcacca
    14701 ctgctgtgcc ttggaatgct agttggagta ataaatctct ggaacagatt tggaatcaca
    14761 cgacctggat ggagtgggac agagaaatta acaattacac aagcttaata cactccttaa
    14821 ttgaagaatc gcaaaaccag caagaaaaga atgaacaaga attattggaa tta
//
