LOCUS       sequence-358012-       15702 bp    DNA     circular SYN 15-DEC-2025
DEFINITION  3rd generation lenti vector encoding dCas9 (S. pyogenes) fused with 
            VP64-p65-Rta (VPR) and 2A puromycin resistance marker; 
            EF1a-dCas9-VPR-P2A-Puro-WPRE).
ACCESSION   .
VERSION     .
KEYWORDS    .
SOURCE      synthetic DNA construct
  ORGANISM  synthetic DNA construct
REFERENCE   1  (bases 1 to 15702)
  AUTHORS   Ho SM, Hartley BJ, Flaherty E, Rajarajan P, Abdelaal R, Obiorah I, 
            Barretto N, Muhammad H, Phatnani HP, Akbarian S, Brennand KJ
  TITLE     Evaluating Synthetic Activation and Repression of 
            Neuropsychiatric-Related Genes in hiPSC-Derived NPCs, Neurons, and 
            Astrocytes.
  JOURNAL   Stem Cell Reports. 2017 Aug 8;9(2):615-628. doi: 
            10.1016/j.stemcr.2017.06.012. Epub 2017 Jul 27.
  PUBMED    28757163
REFERENCE   2  (bases 1 to 15702)
  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: 
            "10.1016/j.stemcr.2017.06.012"; journalName: "Stem Cell Reports"; 
            date: "2017-08-8- 8"; volume: "9"; issue: "2"; pages: "615-628"
COMMENT     Sequence Label: lenti-EF1a-dCas9-VPR-Puro
FEATURES             Location/Qualifiers
     source          1..15702
                     /mol_type="other DNA"
                     /organism="synthetic DNA construct"
     primer_bind     9..30
                     /label=F1ori-F
                     /note="F1 origin, forward primer"
     primer_bind     complement(149..169)
                     /label=pBABE 3'
                     /note="SV40 enhancer, reverse primer for pBABE vectors"
     promoter        154..483
                     /label=SV40 promoter
                     /note="SV40 enhancer and early promoter"
     rep_origin      334..469
                     /label=SV40 ori
                     /note="SV40 origin of replication"
     primer_bind     396..415
                     /label=SV40pro-F
                     /note="SV40 promoter/origin, forward primer"
     promoter        531..578
                     /label=EM7 promoter
                     /note="synthetic bacterial promoter "
     CDS             597..971
                     /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    1101..1222
                     /label=SV40 poly(A) signal
                     /note="SV40 polyadenylation signal"
     primer_bind     complement(1138..1157)
                     /label=SV40pA-R
                     /note="SV40 polyA, reverse primer"
     primer_bind     1192..1211
                     /label=EBV-rev
                     /note="SV40 polyA terminator, reverse primer"
     primer_bind     complement(1271..1287)
                     /label=M13 rev
                     /note="common sequencing primer, one of multiple similar 
                     variants"
     primer_bind     complement(1271..1287)
                     /label=M13 Reverse
                     /note="In lacZ gene. Also called M13-rev"
     primer_bind     complement(1284..1306)
                     /label=M13/pUC Reverse
                     /note="In lacZ gene"
     protein_bind    1295..1311
                     /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(1319..1325,1326..1343,1344..1349))
                     /label=lac promoter
                     /note="promoter for the E. coli lac operon"
     protein_bind    1364..1385
                     /label=CAP binding site
                     /bound_moiety="E. coli catabolite activator protein"
                     /note="CAP binding activates transcription in the presence 
                     of cAMP."
     primer_bind     complement(1502..1519)
                     /label=L4440
                     /note="L4440 vector, forward primer"
     rep_origin      complement(1673..2261)
                     /direction=LEFT
                     /label=ori
                     /note="high-copy-number ColE1/pMB1/pBR322/pUC origin of 
                     replication"
     primer_bind     complement(1753..1772)
                     /label=pBR322ori-F
                     /note="pBR322 origin, forward primer"
     CDS             complement(join(2432..3223,3224..3292))
                     /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     3055..3074
                     /label=Amp-R
                     /note="Ampicillin resistance gene, reverse primer"
     promoter        complement(3293..3397)
                     /gene="bla"
                     /label=AmpR promoter
     primer_bind     complement(3472..3491)
                     /label=pRS-marker
                     /note="pRS vectors, use to sequence yeast selectable 
                     marker"
     enhancer        3663..4042
                     /label=CMV enhancer
                     /note="human cytomegalovirus immediate early enhancer"
     promoter        4044..4242
                     /label=CMV promoter
                     /note="human cytomegalovirus (CMV) immediate early 
                     promoter"
     primer_bind     4197..4217
                     /label=CMV-F
                     /note="Human CMV immediate early promoter, forward primer"
     LTR             4260..4440
                     /label=5' LTR (truncated)
                     /note="truncated 5' long terminal repeat (LTR) from HIV-1"
     misc_feature    4487..4612
                     /label=HIV-1 Psi
                     /note="packaging signal of human immunodeficiency virus 
                     type 1"
     misc_feature    5105..5338
                     /label=RRE
                     /note="The Rev response element (RRE) of HIV-1 allows for 
                     Rev-dependent mRNA export from the nucleus to the 
                     cytoplasm."
     CDS             5523..5567
                     /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    5865..5982
                     /label=cPPT/CTS
                     /note="central polypurine tract and central termination 
                     sequence of HIV-1"
     promoter        6112..7290
                     /label=EF-1-alpha promoter
                     /note="strong constitutive promoter for human elongation 
                     factor EF-1-alpha"
     intron          6343..7281
                     /label=EF-1-alpha intron A
                     /note="intron upstream of the start codon of human 
                     EF-1-alpha"
     primer_bind     7238..7258
                     /label=EF1a-F
                     /note="Human elongation factor-1a promoter, forward primer"
     regulatory      7315..7324
                     /label=Kozak sequence
                     /note="vertebrate consensus sequence for strong initiation 
                     of translation (Kozak, 1987)"
                     /regulatory_class="other"
     CDS             join(7321..7347,7348..7350,7351..9834,9835..9840,9841..9906,
                     9907..9909,9910..11424)
                     /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
                     LYYLQNGRDMYVDQELDINRLSDYDVAAIVPQSFLKDDSIDNKVLTRSDKARGKSDNVP
                     SEEVVKKMKNYWRQLLNAKLITQRKFDNLTKAERGGLSELDKAGFIKRQLVETRQITKH
                     VAQILDSRMNTKYDENDKLIREVKVITLKSKLVSDFRKDFQFYKVREINNYHHAHDAYL
                     NAVVGTALIKKYPKLESEFVYGDYKVYDVRKMIAKSEQEIGKATAKYFFYSNIMNFFKT
                     EITLANGEIRKRPLIETNGETGEIVWDKGRDFATVRKVLSMPQVNIVKKTEVQTGGFSK
                     ESILPKRNSDKLIARKKDWDPKKYGGFDSPTVAYSVLVVAKVEKGKSKKLKSVKELLGI
                     TIMERSSFEKNPIDFLEAKGYKEVKKDLIIKLPKYSLFELENGRKRMLASAGELQKGNE
                     LALPSKYVNFLYLASHYEKLKGSPEDNEQKQLFVEQHKHYLDEIIEQISEFSKRVILAD
                     ANLDKVLSAYNKHRDKPIREQAENIIHLFTLTNLGAPAAFKYFDTTIDRKRYTSTKEVL
                     DATLIHQSITGLYETRIDLSQLGGD"
     CDS             11437..11457
                     /codon_start=1
                     /product="nuclear localization signal of SV40 (simian virus
                     40) large T antigen"
                     /label=SV40 NLS
                     /translation="PKKKRKV"
     protein_bind    11494..11518
                     /gene="mutant version of attB"
                     /label=attB1
                     /bound_moiety="BP Clonase(TM)"
                     /note="recombination site for the Gateway(R) BP reaction"
     CDS             11548..11697
                     /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             11722..11742
                     /codon_start=1
                     /product="nuclear localization signal of SV40 (simian virus
                     40) large T antigen"
                     /label=SV40 NLS
                     /translation="PKKKRKV"
     CDS             12172..12528
                     /codon_start=1
                     /product="transcriptional activation domain of human RelA, 
                     also known as p65 (O'Shea and Perkins, 2008)"
                     /label=RelA (p65) AD
                     /translation="PTQAGEGTLSEALLQLQFDDEDLGALLGNSTDPAVFTDLASVDNS
                     EFQQLLNQGIPVAPHTTEPMLMEYPEAITRLVTGAQRPPDPAPAPLGAPGLPNGLLSGD
                     EDFSSIADMDFSALL"
     CDS             12547..13116
                     /codon_start=1
                     /product="transcriptional activation domain from the human 
                     herpesvirus 4 (Epstein-Barr virus) replication and 
                     transcription activator Rta/BRLF1 (Hardwick et al., 1992; 
                     Chavez et al., 2015)"
                     /label=Rta AD
                     /translation="RDSREGMFLPKPEAGSAISDVFEGREVCQPKRIRPFHPPGSPWAN
                     RPLPASLAPTPTGPVHEPVGSLTPAPVPQPLDPAPAVTPEASHLLEDPDEETSQAVKAL
                     REMADTVIPQKEEAAICGQMDLSHPPPRGHLDELTTTLESMTEDLNLDSPLTPELNEIL
                     DTFLNDECLLHAMHISTGLSIFDTSLF"
     CDS             13132..13188
                     /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             13201..13794
                     /codon_start=1
                     /gene="pac from Streptomyces alboniger"
                     /product="puromycin N-acetyltransferase"
                     /label=PuroR
                     /note="confers resistance to puromycin"
                     /translation="EYKPTVRLATRDDVPRAVRTLAAAFADYPATRHTVDPDRHIERVT
                     ELQELFLTRVGLDIGKVWVADDGAAVAVWTTPESVEAGAVFAEIGPRMAELSGSRLAAQ
                     QQMEGLLAPHRPKEPAWFLATVGVSPDHQGKGLGSAVVLPGVEAAERAGVPAFLETSAP
                     RNLPFYERLGFTVTADVEVPEGPRTWCMTRKPGA"
     primer_bind     13691..13711
                     /label=Puro-F
                     /note="Puromycin resistance gene, forward primer"
     misc_feature    13819..14407
                     /label=WPRE
                     /note="woodchuck hepatitis virus posttranscriptional 
                     regulatory element"
     primer_bind     complement(13872..13892)
                     /label=WPRE-R
                     /note="WPRE, reverse primer"
     CDS             complement(14290..14301)
                     /codon_start=1
                     /product="Factor Xa recognition and cleavage site"
                     /label=Factor Xa site
                     /translation="IEGR"
     primer_bind     complement(14410..14426)
                     /label=KS primer
                     /note="common sequencing primer, one of multiple similar 
                     variants"
     primer_bind     complement(14411..14427)
                     /label=pBluescriptKS
                     /note="For pBluescript vector"
     LTR             14932..15112
                     /label=5' LTR (truncated)
                     /note="truncated 5' long terminal repeat (LTR) from HIV-1"
     primer_bind     complement(15138..15155)
                     /label=BGH-rev
                     /note="Bovine growth hormone terminator, reverse primer. 
                     Also called BGH reverse"
     polyA_signal    15144..15368
                     /label=bGH poly(A) signal
                     /note="bovine growth hormone polyadenylation signal"
     rep_origin      join(15414..15702,1..140)
                     /direction=RIGHT
                     /label=f1 ori
                     /note="f1 bacteriophage origin of replication; arrow 
                     indicates direction of (+) strand synthesis"
     primer_bind     complement(15501..15520)
                     /label=F1ori-R
                     /note="F1 origin, reverse primer"
ORIGIN
        1 ctttaatagt ggactcttgt tccaaactgg aacaacactc aaccctatct cggtctattc
       61 ttttgattta taagggattt tgccgatttc ggcctattgg ttaaaaaatg agctgattta
      121 acaaaaattt aacgcgaatt aattctgtgg aatgtgtgtc agttagggtg tggaaagtcc
      181 ccaggctccc cagcaggcag aagtatgcaa agcatgcatc tcaattagtc agcaaccagg
      241 tgtggaaagt ccccaggctc cccagcaggc agaagtatgc aaagcatgca tctcaattag
      301 tcagcaacca tagtcccgcc cctaactccg cccatcccgc ccctaactcc gcccagttcc
      361 gcccattctc cgccccatgg ctgactaatt ttttttattt atgcagaggc cgaggccgcc
      421 tctgcctctg agctattcca gaagtagtga ggaggctttt ttggaggcct aggcttttgc
      481 aaaaagctcc cgggagcttg tatatccatt ttcggatctg atcagcacgt gttgacaatt
      541 aatcatcggc atagtatatc ggcatagtat aatacgacaa ggtgaggaac taaaccatgg
      601 ccaagttgac cagtgccgtt ccggtgctca ccgcgcgcga cgtcgccgga gcggtcgagt
      661 tctggaccga ccggctcggg ttctcccggg acttcgtgga ggacgacttc gccggtgtgg
      721 tccgggacga cgtgaccctg ttcatcagcg cggtccagga ccaggtggtg ccggacaaca
      781 ccctggcctg ggtgtgggtg cgcggcctgg acgagctgta cgccgagtgg tcggaggtcg
      841 tgtccacgaa cttccgggac gcctccgggc cggccatgac cgagatcggc gagcagccgt
      901 gggggcggga gttcgccctg cgcgacccgg ccggcaactg cgtgcacttc gtggccgagg
      961 agcaggactg acacgtgcta cgagatttcg attccaccgc cgccttctat gaaaggttgg
     1021 gcttcggaat cgttttccgg gacgccggct ggatgatcct ccagcgcggg gatctcatgc
     1081 tggagttctt cgcccacccc aacttgttta ttgcagctta taatggttac aaataaagca
     1141 atagcatcac aaatttcaca aataaagcat ttttttcact gcattctagt tgtggtttgt
     1201 ccaaactcat caatgtatct tatcatgtct gtataccgtc gacctctagc tagagcttgg
     1261 cgtaatcatg gtcatagctg tttcctgtgt gaaattgtta tccgctcaca attccacaca
     1321 acatacgagc cggaagcata aagtgtaaag cctggggtgc ctaatgagtg agctaactca
     1381 cattaattgc gttgcgctca ctgcccgctt tccagtcggg aaacctgtcg tgccagctgc
     1441 attaatgaat cggccaacgc gcggggagag gcggtttgcg tattgggcgc tcttccgctt
     1501 cctcgctcac tgactcgctg cgctcggtcg ttcggctgcg gcgagcggta tcagctcact
     1561 caaaggcggt aatacggtta tccacagaat caggggataa cgcaggaaag aacatgtgag
     1621 caaaaggcca gcaaaaggcc aggaaccgta aaaaggccgc gttgctggcg tttttccata
     1681 ggctccgccc ccctgacgag catcacaaaa atcgacgctc aagtcagagg tggcgaaacc
     1741 cgacaggact ataaagatac caggcgtttc cccctggaag ctccctcgtg cgctctcctg
     1801 ttccgaccct gccgcttacc ggatacctgt ccgcctttct cccttcggga agcgtggcgc
     1861 tttctcatag ctcacgctgt aggtatctca gttcggtgta ggtcgttcgc tccaagctgg
     1921 gctgtgtgca cgaacccccc gttcagcccg accgctgcgc cttatccggt aactatcgtc
     1981 ttgagtccaa cccggtaaga cacgacttat cgccactggc agcagccact ggtaacagga
     2041 ttagcagagc gaggtatgta ggcggtgcta cagagttctt gaagtggtgg cctaactacg
     2101 gctacactag aagaacagta tttggtatct gcgctctgct gaagccagtt accttcggaa
     2161 aaagagttgg tagctcttga tccggcaaac aaaccaccgc tggtagcggt ggtttttttg
     2221 tttgcaagca gcagattacg cgcagaaaaa aaggatctca agaagatcct ttgatctttt
     2281 ctacggggtc tgacgctcag tggaacgaaa actcacgtta agggattttg gtcatgagat
     2341 tatcaaaaag gatcttcacc tagatccttt taaattaaaa atgaagtttt aaatcaatct
     2401 aaagtatata tgagtaaact tggtctgaca gttaccaatg cttaatcagt gaggcaccta
     2461 tctcagcgat ctgtctattt cgttcatcca tagttgcctg actccccgtc gtgtagataa
     2521 ctacgatacg ggagggctta ccatctggcc ccagtgctgc aatgataccg cgagacccac
     2581 gctcaccggc tccagattta tcagcaataa accagccagc cggaagggcc gagcgcagaa
     2641 gtggtcctgc aactttatcc gcctccatcc agtctattaa ttgttgccgg gaagctagag
     2701 taagtagttc gccagttaat agtttgcgca acgttgttgc cattgctaca ggcatcgtgg
     2761 tgtcacgctc gtcgtttggt atggcttcat tcagctccgg ttcccaacga tcaaggcgag
     2821 ttacatgatc ccccatgttg tgcaaaaaag cggttagctc cttcggtcct ccgatcgttg
     2881 tcagaagtaa gttggccgca gtgttatcac tcatggttat ggcagcactg cataattctc
     2941 ttactgtcat gccatccgta agatgctttt ctgtgactgg tgagtactca accaagtcat
     3001 tctgagaata gtgtatgcgg cgaccgagtt gctcttgccc ggcgtcaata cgggataata
     3061 ccgcgccaca tagcagaact ttaaaagtgc tcatcattgg aaaacgttct tcggggcgaa
     3121 aactctcaag gatcttaccg ctgttgagat ccagttcgat gtaacccact cgtgcaccca
     3181 actgatcttc agcatctttt actttcacca gcgtttctgg gtgagcaaaa acaggaaggc
     3241 aaaatgccgc aaaaaaggga ataagggcga cacggaaatg ttgaatactc atactcttcc
     3301 tttttcaata ttattgaagc atttatcagg gttattgtct catgagcgga tacatatttg
     3361 aatgtattta gaaaaataaa caaatagggg ttccgcgcac atttccccga aaagtgccac
     3421 ctgacgtcga cggatcggga gatctcccga tcccctatgg tgcactctca gtacaatctg
     3481 ctctgatgcc gcatagttaa gccagtatct gctccctgct tgtgtgttgg aggtcgctga
     3541 gtagtgcgcg agcaaaattt aagctacaac aaggcaaggc ttgaccgaca attgcatgaa
     3601 gaatctgctt agggttaggc gttttgcgct gcttcgcgat gtacgggcca gatatacgcg
     3661 ttgacattga ttattgacta gttattaata gtaatcaatt acggggtcat tagttcatag
     3721 cccatatatg gagttccgcg ttacataact tacggtaaat ggcccgcctg gctgaccgcc
     3781 caacgacccc cgcccattga cgtcaataat gacgtatgtt cccatagtaa cgccaatagg
     3841 gactttccat tgacgtcaat gggtggagta tttacggtaa actgcccact tggcagtaca
     3901 tcaagtgtat catatgccaa gtacgccccc tattgacgtc aatgacggta aatggcccgc
     3961 ctggcattat gcccagtaca tgaccttatg ggactttcct acttggcagt acatctacgt
     4021 attagtcatc gctattacca tggtgatgcg gttttggcag tacatcaatg ggcgtggata
     4081 gcggtttgac tcacggggat ttccaagtct ccaccccatt gacgtcaatg ggagtttgtt
     4141 ttggcaccaa aatcaacggg actttccaaa atgtcgtaac aactccgccc cattgacgca
     4201 aatgggcggt aggcgtgtac ggtgggaggt ctatataagc agcgcgtttt gcctgtactg
     4261 ggtctctctg gttagaccag atctgagcct gggagctctc tggctaacta gggaacccac
     4321 tgcttaagcc tcaataaagc ttgccttgag tgcttcaagt agtgtgtgcc cgtctgttgt
     4381 gtgactctgg taactagaga tccctcagac ccttttagtc agtgtggaaa atctctagca
     4441 gtggcgcccg aacagggact tgaaagcgaa agggaaacca gaggagctct ctcgacgcag
     4501 gactcggctt gctgaagcgc gcacggcaag aggcgagggg cggcgactgg tgagtacgcc
     4561 aaaaattttg actagcggag gctagaagga gagagatggg tgcgagagcg tcagtattaa
     4621 gcgggggaga attagatcgc gatgggaaaa aattcggtta aggccagggg gaaagaaaaa
     4681 atataaatta aaacatatag tatgggcaag cagggagcta gaacgattcg cagttaatcc
     4741 tggcctgtta gaaacatcag aaggctgtag acaaatactg ggacagctac aaccatccct
     4801 tcagacagga tcagaagaac ttagatcatt atataataca gtagcaaccc tctattgtgt
     4861 gcatcaaagg atagagataa aagacaccaa ggaagcttta gacaagatag aggaagagca
     4921 aaacaaaagt aagaccaccg cacagcaagc ggccgctgat cttcagacct ggaggaggag
     4981 atatgaggga caattggaga agtgaattat ataaatataa agtagtaaaa attgaaccat
     5041 taggagtagc acccaccaag gcaaagagaa gagtggtgca gagagaaaaa agagcagtgg
     5101 gaataggagc tttgttcctt gggttcttgg gagcagcagg aagcactatg ggcgcagcgt
     5161 caatgacgct gacggtacag gccagacaat tattgtctgg tatagtgcag cagcagaaca
     5221 atttgctgag ggctattgag gcgcaacagc atctgttgca actcacagtc tggggcatca
     5281 agcagctcca ggcaagaatc ctggctgtgg aaagatacct aaaggatcaa cagctcctgg
     5341 ggatttgggg ttgctctgga aaactcattt gcaccactgc tgtgccttgg aatgctagtt
     5401 ggagtaataa atctctggaa cagatttgga atcacacgac ctggatggag tgggacagag
     5461 aaattaacaa ttacacaagc ttaatacact ccttaattga agaatcgcaa aaccagcaag
     5521 aaaagaatga acaagaatta ttggaattag ataaatgggc aagtttgtgg aattggttta
     5581 acataacaaa ttggctgtgg tatataaaat tattcataat gatagtagga ggcttggtag
     5641 gtttaagaat agtttttgct gtactttcta tagtgaatag agttaggcag ggatattcac
     5701 cattatcgtt tcagacccac ctcccaaccc cgaggggacc cgacaggccc gaaggaatag
     5761 aagaagaagg tggagagaga gacagagaca gatccattcg attagtgaac ggatcggcac
     5821 tgcgtgcgcc aattctgcag acaaatggca gtattcatcc acaattttaa aagaaaaggg
     5881 gggattgggg ggtacagtgc aggggaaaga atagtagaca taatagcaac agacatacaa
     5941 actaaagaat tacaaaaaca aattacaaaa attcaaaatt ttcgggttta ttacagggac
     6001 agcagagatc cagtttggtt aattagctag ctgcaaagat ggataaagtt ttaaacagag
     6061 aggaatcttt gcagctaatg gaccttctag gtcttgaaag gagtgggaat tggctccggt
     6121 gcccgtcagt gggcagagcg cacatcgccc acagtccccg agaagttggg gggaggggtc
     6181 ggcaattgaa ccggtgccta gagaaggtgg cgcggggtaa actgggaaag tgatgtcgtg
     6241 tactggctcc gcctttttcc cgagggtggg ggagaaccgt atataagtgc agtagtcgcc
     6301 gtgaacgttc tttttcgcaa cgggtttgcc gccagaacac aggtaagtgc cgtgtgtggt
     6361 tcccgcgggc ctggcctctt tacgggttat ggcccttgcg tgccttgaat tacttccacc
     6421 tggctgcagt acgtgattct tgatcccgag cttcgggttg gaagtgggtg ggagagttcg
     6481 aggccttgcg cttaaggagc cccttcgcct cgtgcttgag ttgaggcctg gcctgggcgc
     6541 tggggccgcc gcgtgcgaat ctggtggcac cttcgcgcct gtctcgctgc tttcgataag
     6601 tctctagcca tttaaaattt ttgatgacct gctgcgacgc tttttttctg gcaagatagt
     6661 cttgtaaatg cgggccaaga tctgcacact ggtatttcgg tttttggggc cgcgggcggc
     6721 gacggggccc gtgcgtccca gcgcacatgt tcggcgaggc ggggcctgcg agcgcggcca
     6781 ccgagaatcg gacgggggta gtctcaagct ggccggcctg ctctggtgcc tggcctcgcg
     6841 ccgccgtgta tcgccccgcc ctgggcggca aggctggccc ggtcggcacc agttgcgtga
     6901 gcggaaagat ggccgcttcc cggccctgct gcagggagct caaaatggag gacgcggcgc
     6961 tcgggagagc gggcgggtga gtcacccaca caaaggaaaa gggcctttcc gtcctcagcc
     7021 gtcgcttcat gtgactccac ggagtaccgg gcgccgtcca ggcacctcga ttagttctcg
     7081 agcttttgga gtacgtcgtc tttaggttgg ggggaggggt tttatgcgat ggagtttccc
     7141 cacactgagt gggtggagac tgaagttagg ccagcttggc acttgatgta attctccttg
     7201 gaatttgccc tttttgagtt tggatcttgg ttcattctca agcctcagac agtggttcaa
     7261 agtttttttc ttccatttca ggtgtcgtga cgtacgctag aggatcgaac ccttgccacc
     7321 atggacaaga agtactccat tgggctcgct atcggcacaa acagcgtcgg ctgggccgtc
     7381 attacggacg agtacaaggt gccgagcaaa aaattcaaag ttctgggcaa taccgatcgc
     7441 cacagcataa agaagaacct cattggcgcc ctcctgttcg actccgggga gacggccgaa
     7501 gccacgcggc tcaaaagaac agcacggcgc agatataccc gcagaaagaa tcggatctgc
     7561 tacctgcagg agatctttag taatgagatg gctaaggtgg atgactcttt cttccatagg
     7621 ctggaggagt cctttttggt ggaggaggat aaaaagcacg agcgccaccc aatctttggc
     7681 aatatcgtgg acgaggtggc gtaccatgaa aagtacccaa ccatatatca tctgaggaag
     7741 aagcttgtag acagtactga taaggctgac ttgcggttga tctatctcgc gctggcgcat
     7801 atgatcaaat ttcggggaca cttcctcatc gagggggacc tgaacccaga caacagcgat
     7861 gtcgacaaac tctttatcca actggttcag acttacaatc agcttttcga agagaacccg
     7921 atcaacgcat ccggagttga cgccaaagca atcctgagcg ctaggctgtc caaatcccgg
     7981 cggctcgaaa acctcatcgc acagctccct ggggagaaga agaacggcct gtttggtaat
     8041 cttatcgccc tgtcactcgg gctgaccccc aactttaaat ctaacttcga cctggccgaa
     8101 gatgccaagc ttcaactgag caaagacacc tacgatgatg atctcgacaa tctgctggcc
     8161 cagatcggcg accagtacgc agaccttttt ttggcggcaa agaacctgtc agacgccatt
     8221 ctgctgagtg atattctgcg agtgaacacg gagatcacca aagctccgct gagcgctagt
     8281 atgatcaagc gctatgatga gcaccaccaa gacttgactt tgctgaaggc ccttgtcaga
     8341 cagcaactgc ctgagaagta caaggaaatt ttcttcgatc agtctaaaaa tggctacgcc
     8401 ggatacattg acggcggagc aagccaggag gaattttaca aatttattaa gcccatcttg
     8461 gaaaaaatgg acggcaccga ggagctgctg gtaaagctta acagagaaga tctgttgcgc
     8521 aaacagcgca ctttcgacaa tggaagcatc ccccaccaga ttcacctggg cgaactgcac
     8581 gctatcctca ggcggcaaga ggatttctac ccctttttga aagataacag ggaaaagatt
     8641 gagaaaatcc tcacatttcg gataccctac tatgtaggcc ccctcgcccg gggaaattcc
     8701 agattcgcgt ggatgactcg caaatcagaa gagaccatca ctccctggaa cttcgaggaa
     8761 gtcgtggata agggggcctc tgcccagtcc ttcatcgaaa ggatgactaa ctttgataaa
     8821 aatctgccta acgaaaaggt gcttcctaaa cactctctgc tgtacgagta cttcacagtt
     8881 tataacgagc tcaccaaggt caaatacgtc acagaaggga tgagaaagcc agcattcctg
     8941 tctggagagc agaagaaagc tatcgtggac ctcctcttca agacgaaccg gaaagttacc
     9001 gtgaaacagc tcaaagaaga ctatttcaaa aagattgaat gtttcgactc tgttgaaatc
     9061 agcggagtgg aggatcgctt caacgcatcc ctgggaacgt atcacgatct cctgaaaatc
     9121 attaaagaca aggacttcct ggacaatgag gagaacgagg acattcttga ggacattgtc
     9181 ctcaccctta cgttgtttga agatagggag atgattgaag aacgcttgaa aacttacgct
     9241 catctcttcg acgacaaagt catgaaacag ctcaagaggc gccgatatac aggatggggg
     9301 cggctgtcaa gaaaactgat caatgggatc cgagacaagc agagtggaaa gacaatcctg
     9361 gattttctta agtccgatgg atttgccaac cggaacttca tgcagttgat ccatgatgac
     9421 tctctcacct ttaaggagga catccagaaa gcacaagttt ctggccaggg ggacagtctt
     9481 cacgagcaca tcgctaatct tgcaggtagc ccagctatca aaaagggaat actgcagacc
     9541 gttaaggtcg tggatgaact cgtcaaagta atgggaaggc ataagcccga gaatatcgtt
     9601 atcgagatgg cccgagagaa ccaaactacc cagaagggac agaagaacag tagggaaagg
     9661 atgaagagga ttgaagaggg tataaaagaa ctggggtccc aaatccttaa ggaacaccca
     9721 gttgaaaaca cccagcttca gaatgagaag ctctacctgt actacctgca gaacggcagg
     9781 gacatgtacg tggatcagga actggacatc aatcggctct ccgactacga cgtggctgct
     9841 atcgtgcccc agtcttttct caaagatgat tctattgata ataaagtgtt gacaagatcc
     9901 gataaagcta gagggaagag tgataacgtc ccctcagaag aagttgtcaa gaaaatgaaa
     9961 aattattggc ggcagctgct gaacgccaaa ctgatcacac aacggaagtt cgataatctg
    10021 actaaggctg aacgaggtgg cctgtctgag ttggataaag ccggcttcat caaaaggcag
    10081 cttgttgaga cacgccagat caccaagcac gtggcccaaa ttctcgattc acgcatgaac
    10141 accaagtacg atgaaaatga caaactgatt cgagaggtga aagttattac tctgaagtct
    10201 aagctggtct cagatttcag aaaggacttt cagttttata aggtgagaga gatcaacaat
    10261 taccaccatg cgcatgatgc ctacctgaat gcagtggtag gcactgcact tatcaaaaaa
    10321 tatcccaagc ttgaatctga atttgtttac ggagactata aagtgtacga tgttaggaaa
    10381 atgatcgcaa agtctgagca ggaaataggc aaggccaccg ctaagtactt cttttacagc
    10441 aatattatga attttttcaa gaccgagatt acactggcca atggagagat tcggaagcga
    10501 ccacttatcg aaacaaacgg agaaacagga gaaatcgtgt gggacaaggg tagggatttc
    10561 gcgacagtcc ggaaggtcct gtccatgccg caggtgaaca tcgttaaaaa gaccgaagta
    10621 cagaccggag gcttctccaa ggaaagtatc ctcccgaaaa ggaacagcga caagctgatc
    10681 gcacgcaaaa aagattggga ccccaagaaa tacggcggat tcgattctcc tacagtcgct
    10741 tacagtgtac tggttgtggc caaagtggag aaagggaagt ctaaaaaact caaaagcgtc
    10801 aaggaactgc tgggcatcac aatcatggag cgatcaagct tcgaaaaaaa ccccatcgac
    10861 tttctcgagg cgaaaggata taaagaggtc aaaaaagacc tcatcattaa gcttcccaag
    10921 tactctctct ttgagcttga aaacggccgg aaacgaatgc tcgctagtgc gggcgagctg
    10981 cagaaaggta acgagctggc actgccctct aaatacgtta atttcttgta tctggccagc
    11041 cactatgaaa agctcaaagg gtctcccgaa gataatgagc agaagcagct gttcgtggaa
    11101 caacacaaac actaccttga tgagatcatc gagcaaataa gcgaattctc caaaagagtg
    11161 atcctcgccg acgctaacct cgataaggtg ctttctgctt acaataagca cagggataag
    11221 cccatcaggg agcaggcaga aaacattatc cacttgttta ctctgaccaa cttgggcgcg
    11281 cctgcagcct tcaagtactt cgacaccacc atagacagaa agcggtacac ctctacaaag
    11341 gaggtcctgg acgccacact gattcatcag tcaattacgg ggctctatga aacaagaatc
    11401 gacctctctc agctcggtgg agacagcagg gctgacccca agaagaagag gaaggtgtcg
    11461 ccagggatcc gtcgacttga cgcgttgata tcaacaagtt tgtacaaaaa agcaggctac
    11521 aaagaggcca gcggttccgg acgggctgac gcattggacg attttgatct ggatatgctg
    11581 ggaagtgacg ccctcgatga ttttgacctt gacatgcttg gttcggatgc ccttgatgac
    11641 tttgacctcg acatgctcgg cagtgacgcc cttgatgatt tcgacctgga catgctgatt
    11701 aactctagaa gttccggatc tccgaaaaag aaacgcaaag ttggtagcca gtacctgccc
    11761 gacaccgacg accggcaccg gatcgaggaa aagcggaagc ggacctacga gacattcaag
    11821 agcatcatga agaagtcccc cttcagcggc cccaccgacc ctagacctcc acctagaaga
    11881 atcgccgtgc ccagcagatc cagcgccagc gtgccaaaac ctgcccccca gccttacccc
    11941 ttcaccagca gcctgagcac catcaactac gacgagttcc ctaccatggt gttccccagc
    12001 ggccagatct ctcaggcctc tgctctggct ccagcccctc ctcaggtgct gcctcaggct
    12061 cctgctcctg caccagctcc agccatggtg tctgcactgg ctcaggcacc agcacccgtg
    12121 cctgtgctgg ctcctggacc tccacaggct gtggctccac cagcccctaa acctacacag
    12181 gccggcgagg gcacactgtc tgaagctctg ctgcagctgc agttcgacga cgaggatctg
    12241 ggagccctgc tgggaaacag caccgatcct gccgtgttca ccgacctggc cagcgtggac
    12301 aacagcgagt tccagcagct gctgaaccag ggcatccctg tggcccctca caccaccgag
    12361 cccatgctga tggaataccc cgaggccatc acccggctcg tgacaggcgc tcagaggcct
    12421 cctgatccag ctcctgcccc tctgggagca ccaggcctgc ctaatggact gctgtctggc
    12481 gacgaggact tcagctctat cgccgatatg gatttctcag ccttgctggg ctctggcagc
    12541 ggcagccggg attccaggga agggatgttt ttgccgaagc ctgaggccgg ctccgctatt
    12601 agtgacgtgt ttgagggccg cgaggtgtgc cagccaaaac gaatccggcc atttcatcct
    12661 ccaggaagtc catgggccaa ccgcccactc cccgccagcc tcgcaccaac accaaccggt
    12721 ccagtacatg agccagtcgg gtcactgacc ccggcaccag tccctcagcc actggatcca
    12781 gcgcccgcag tgactcccga ggccagtcac ctgttggagg atcccgatga agagacgagc
    12841 caggctgtca aagcccttcg ggagatggcc gatactgtga ttccccagaa ggaagaggct
    12901 gcaatctgtg gccaaatgga cctttcccat ccgcccccaa ggggccatct ggatgagctg
    12961 acaaccacac ttgagtccat gaccgaggat ctgaacctgg actcacccct gaccccggaa
    13021 ttgaacgaga ttctggatac cttcctgaac gacgagtgcc tcttgcatgc catgcatatc
    13081 agcacaggac tgtccatctt cgacacatct ctctttgaat tcggatctgg tgccacgaat
    13141 ttctcgctac tcaagcaggc cggtgatgtc gaggaaaacc ctggtcctat ggtgtctaga
    13201 gagtacaagc ccacggtgcg cctcgccacc cgcgacgacg tccccagggc cgtacgcacc
    13261 ctcgccgccg cgttcgccga ctaccccgcc acgcgccaca ccgtcgatcc ggaccgccac
    13321 atcgagcggg tcaccgagct gcaagaactc ttcctcacgc gcgtcgggct cgacatcggc
    13381 aaggtgtggg tcgcggacga cggcgccgcg gtggcggtct ggaccacgcc ggagagcgtc
    13441 gaagcggggg cggtgttcgc cgagatcggc ccgcgcatgg ccgagttgag cggttcccgg
    13501 ctggccgcgc agcaacagat ggaaggcctc ctggcgccgc accggcccaa ggagcccgcg
    13561 tggttcctgg ccaccgtcgg cgtctcgccc gaccaccagg gcaagggtct gggcagcgcc
    13621 gtcgtgctcc ccggagtgga ggcggccgag cgcgccgggg tgcccgcctt cctggagacc
    13681 tccgcgcccc gcaacctccc cttctacgag cggctcggct tcaccgtcac cgccgacgtc
    13741 gaggtgcccg aaggaccgcg cacctggtgc atgacccgca agcccggtgc ctgagaattc
    13801 gatatcaagc ttatcggtaa tcaacctctg gattacaaaa tttgtgaaag attgactggt
    13861 attcttaact atgttgctcc ttttacgcta tgtggatacg ctgctttaat gcctttgtat
    13921 catgctattg cttcccgtat ggctttcatt ttctcctcct tgtataaatc ctggttgctg
    13981 tctctttatg aggagttgtg gcccgttgtc aggcaacgtg gcgtggtgtg cactgtgttt
    14041 gctgacgcaa cccccactgg ttggggcatt gccaccacct gtcagctcct ttccgggact
    14101 ttcgctttcc ccctccctat tgccacggcg gaactcatcg ccgcctgcct tgcccgctgc
    14161 tggacagggg ctcggctgtt gggcactgac aattccgtgg tgttgtcggg gaaatcatcg
    14221 tcctttcctt ggctgctcgc ctgtgttgcc acctggattc tgcgcgggac gtccttctgc
    14281 tacgtccctt cggccctcaa tccagcggac cttccttccc gcggcctgct gccggctctg
    14341 cggcctcttc cgcgtcttcg ccttcgccct cagacgagtc ggatctccct ttgggccgcc
    14401 tccccgcatc gataccgtcg acctcgagac ctagaaaaac atggagcaat cacaagtagc
    14461 aatacagcag ctaccaatgc tgattgtgcc tggctagaag cacaagagga ggaggaggtg
    14521 ggttttccag tcacacctca ggtaccttta agaccaatga cttacaaggc agctgtagat
    14581 cttagccact ttttaaaaga aaagggggga ctggaagggc taattcactc ccaacgaaga
    14641 caagatatcc ttgatctgtg gatctaccac acacaaggct acttccctga ttggcagaac
    14701 tacacaccag ggccagggat cagatatcca ctgacctttg gatggtgcta caagctagta
    14761 ccagttgagc aagagaaggt agaagaagcc aatgaaggag agaacacccg cttgttacac
    14821 cctgtgagcc tgcatgggat ggatgacccg gagagagaag tattagagtg gaggtttgac
    14881 agccgcctag catttcatca catggcccga gagctgcatc cggactgtac tgggtctctc
    14941 tggttagacc agatctgagc ctgggagctc tctggctaac tagggaaccc actgcttaag
    15001 cctcaataaa gcttgccttg agtgcttcaa gtagtgtgtg cccgtctgtt gtgtgactct
    15061 ggtaactaga gatccctcag acccttttag tcagtgtgga aaatctctag cagggcccgt
    15121 ttaaacccgc tgatcagcct cgactgtgcc ttctagttgc cagccatctg ttgtttgccc
    15181 ctcccccgtg ccttccttga ccctggaagg tgccactccc actgtccttt cctaataaaa
    15241 tgaggaaatt gcatcgcatt gtctgagtag gtgtcattct attctggggg gtggggtggg
    15301 gcaggacagc aagggggagg attgggaaga caatagcagg catgctgggg atgcggtggg
    15361 ctctatggct tctgaggcgg aaagaaccag ctggggctct agggggtatc cccacgcgcc
    15421 ctgtagcggc gcattaagcg cggcgggtgt ggtggttacg cgcagcgtga ccgctacact
    15481 tgccagcgcc ctagcgcccg ctcctttcgc tttcttccct tcctttctcg ccacgttcgc
    15541 cggctttccc cgtcaagctc taaatcgggg gctcccttta gggttccgat ttagtgcttt
    15601 acggcacctc gaccccaaaa aacttgatta gggtgatggt tcacgtagtg ggccatcgcc
    15661 ctgatagacg gtttttcgcc ctttgacgtt ggagtccacg tt
//
