LOCUS       sequence-369755-       14997 bp    DNA     circular SYN 15-APR-2025
DEFINITION  dCas9 effector fusion for manipulating transcription.
ACCESSION   .
VERSION     .
KEYWORDS    .
SOURCE      synthetic DNA construct
  ORGANISM  synthetic DNA construct
REFERENCE   1  (bases 1 to 14997)
  AUTHORS   Tycko J, Van MV, Aradhana, DelRosso N, Ye H, Yao D, Valbuena R, 
            Vaughan-Jackson A, Xu X, Ludwig C, Spees K, Liu K, Gu M, Khare V, 
            Mukund AX, Suzuki PH, Arana S, Zhang C, Du PP, Ornstein TS, Hess GT,
            Kamber RA, Qi LS, Khalil AS, Bintu L, Bassik MC
  TITLE     Development of compact transcriptional effectors using 
            high-throughput measurements in diverse contexts.
  JOURNAL   Nat Biotechnol. 2024 Nov 1. doi: 10.1038/s41587-024-02442-6.
  PUBMED    39487265
REFERENCE   2  (bases 1 to 14997)
  AUTHORS   .
  TITLE     Direct Submission
  JOURNAL   Exported Apr 15, 2025 from SnapGene Server 8.0.1
            https://www.snapgene.com
COMMENT     SGRef: number: 1; type: "Journal Article"; journalName: "Nat 
            Biotechnol. 2024 Nov 1. doi: 10.1038/s41587-024-02442-6."
COMMENT     Sequence Label: pJT241 Lenti pEF-dCas9-3XFLAG-WT ZNF10
            KRAB-T2...
FEATURES             Location/Qualifiers
     source          1..14997
                     /mol_type="other DNA"
                     /organism="synthetic DNA construct"
     primer_bind     complement(13..33)
                     /label=pBABE 3'
                     /note="SV40 enhancer, reverse primer for pBABE vectors"
     promoter        18..347
                     /label=SV40 promoter
                     /note="SV40 enhancer and early promoter"
     rep_origin      198..333
                     /label=SV40 ori
                     /note="SV40 origin of replication"
     primer_bind     260..279
                     /label=SV40pro-F
                     /note="SV40 promoter/origin, forward primer"
     promoter        395..442
                     /label=EM7 promoter
                     /note="synthetic bacterial promoter "
     CDS             461..835
                     /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    965..1086
                     /label=SV40 poly(A) signal
                     /note="SV40 polyadenylation signal"
     primer_bind     complement(1002..1021)
                     /label=SV40pA-R
                     /note="SV40 polyA, reverse primer"
     primer_bind     1056..1075
                     /label=EBV-rev
                     /note="SV40 polyA terminator, reverse primer"
     primer_bind     complement(1135..1151)
                     /label=M13 rev
                     /note="common sequencing primer, one of multiple similar 
                     variants"
     primer_bind     complement(1135..1151)
                     /label=M13 Reverse
                     /note="In lacZ gene. Also called M13-rev"
     primer_bind     complement(1148..1170)
                     /label=M13/pUC Reverse
                     /note="In lacZ gene"
     protein_bind    1159..1175
                     /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(1183..1189,1190..1207,1208..1213))
                     /label=lac promoter
                     /note="promoter for the E. coli lac operon"
     protein_bind    1228..1249
                     /label=CAP binding site
                     /bound_moiety="E. coli catabolite activator protein"
                     /note="CAP binding activates transcription in the presence 
                     of cAMP."
     primer_bind     complement(1366..1383)
                     /label=L4440
                     /note="L4440 vector, forward primer"
     rep_origin      complement(1537..2125)
                     /direction=LEFT
                     /label=ori
                     /note="high-copy-number ColE1/pMB1/pBR322/pUC origin of 
                     replication"
     primer_bind     complement(1617..1636)
                     /label=pBR322ori-F
                     /note="pBR322 origin, forward primer"
     CDS             complement(join(2296..3087,3088..3156))
                     /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     2919..2938
                     /label=Amp-R
                     /note="Ampicillin resistance gene, reverse primer"
     promoter        complement(3157..3261)
                     /gene="bla"
                     /label=AmpR promoter
     primer_bind     complement(3336..3355)
                     /label=pRS-marker
                     /note="pRS vectors, use to sequence yeast selectable 
                     marker"
     enhancer        3527..3906
                     /label=CMV enhancer
                     /note="human cytomegalovirus immediate early enhancer"
     promoter        3908..4106
                     /label=CMV promoter
                     /note="human cytomegalovirus (CMV) immediate early 
                     promoter"
     primer_bind     4061..4081
                     /label=CMV-F
                     /note="Human CMV immediate early promoter, forward primer"
     LTR             4124..4304
                     /label=5' LTR (truncated)
                     /note="truncated 5' long terminal repeat (LTR) from HIV-1"
     misc_feature    4351..4476
                     /label=HIV-1 Psi
                     /note="packaging signal of human immunodeficiency virus 
                     type 1"
     misc_feature    4969..5202
                     /label=RRE
                     /note="The Rev response element (RRE) of HIV-1 allows for 
                     Rev-dependent mRNA export from the nucleus to the 
                     cytoplasm."
     CDS             5387..5431
                     /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    5729..5846
                     /label=cPPT/CTS
                     /note="central polypurine tract and central termination 
                     sequence of HIV-1"
     promoter        5976..7154
                     /label=EF-1-alpha promoter
                     /note="strong constitutive promoter for human elongation 
                     factor EF-1-alpha"
     intron          6207..7145
                     /label=EF-1-alpha intron A
                     /note="intron upstream of the start codon of human 
                     EF-1-alpha"
     primer_bind     7102..7122
                     /label=EF1a-F
                     /note="Human elongation factor-1a promoter, forward primer"
     CDS             7170..7217
                     /codon_start=1
                     /product="bipartite nuclear localization signal from 
                     nucleoplasmin"
                     /label=nucleoplasmin NLS
                     /translation="KRPAATKKAGQAKKKK"
     CDS             join(7218..7241,7242..7244,7245..9800,9801..9803,
                     9804..11318)
                     /codon_start=1
                     /product="catalytically dead mutant of the Cas9 
                     endonuclease from the Streptococcus pyogenes Type II 
                     CRISPR/Cas system"
                     /label=dCas9
                     /note="RNA-guided DNA-binding protein that lacks 
                     endonuclease activity due to the D10A mutation in the RuvC 
                     catalytic domain and the N863A mutation in the HNH 
                     catalytic domain"
                     /translation="DKKYSIGLAIGTNSVGWAVITDEYKVPSKKFKVLGNTDRHSIKKN
                     LIGALLFDSGETAEATRLKRTARRRYTRRKNRICYLQEIFSNEMAKVDDSFFHRLEESF
                     LVEEDKKHERHPIFGNIVDEVAYHEKYPTIYHLRKKLVDSTDKADLRLIYLALAHMIKF
                     RGHFLIEGDLNPDNSDVDKLFIQLVQTYNQLFEENPINASGVDAKAILSARLSKSRRLE
                     NLIAQLPGEKKNGLFGNLIALSLGLTPNFKSNFDLAEDAKLQLSKDTYDDDLDNLLAQI
                     GDQYADLFLAAKNLSDAILLSDILRVNTEITKAPLSASMIKRYDEHHQDLTLLKALVRQ
                     QLPEKYKEIFFDQSKNGYAGYIDGGASQEEFYKFIKPILEKMDGTEELLVKLNREDLLR
                     KQRTFDNGSIPHQIHLGELHAILRRQEDFYPFLKDNREKIEKILTFRIPYYVGPLARGN
                     SRFAWMTRKSEETITPWNFEEVVDKGASAQSFIERMTNFDKNLPNEKVLPKHSLLYEYF
                     TVYNELTKVKYVTEGMRKPAFLSGEQKKAIVDLLFKTNRKVTVKQLKEDYFKKIECFDS
                     VEISGVEDRFNASLGTYHDLLKIIKDKDFLDNEENEDILEDIVLTLTLFEDREMIEERL
                     KTYAHLFDDKVMKQLKRRRYTGWGRLSRKLINGIRDKQSGKTILDFLKSDGFANRNFMQ
                     LIHDDSLTFKEDIQKAQVSGQGDSLHEHIANLAGSPAIKKGILQTVKVVDELVKVMGRH
                     KPENIVIEMARENQTTQKGQKNSRERMKRIEEGIKELGSQILKEHPVENTQLQNEKLYL
                     YYLQNGRDMYVDQELDINRLSDYDVDHIVPQSFLKDDSIDNKVLTRSDKARGKSDNVPS
                     EEVVKKMKNYWRQLLNAKLITQRKFDNLTKAERGGLSELDKAGFIKRQLVETRQITKHV
                     AQILDSRMNTKYDENDKLIREVKVITLKSKLVSDFRKDFQFYKVREINNYHHAHDAYLN
                     AVVGTALIKKYPKLESEFVYGDYKVYDVRKMIAKSEQEIGKATAKYFFYSNIMNFFKTE
                     ITLANGEIRKRPLIETNGETGEIVWDKGRDFATVRKVLSMPQVNIVKKTEVQTGGFSKE
                     SILPKRNSDKLIARKKDWDPKKYGGFDSPTVAYSVLVVAKVEKGKSKKLKSVKELLGIT
                     IMERSSFEKNPIDFLEAKGYKEVKKDLIIKLPKYSLFELENGRKRMLASAGELQKGNEL
                     ALPSKYVNFLYLASHYEKLKGSPEDNEQKQLFVEQHKHYLDEIIEQISEFSKRVILADA
                     NLDKVLSAYNKHRDKPIREQAENIIHLFTLTNLGAPAAFKYFDTTIDRKRYTSTKEVLD
                     ATLIHQSITGLYETRIDLSQLGGD"
     CDS             11373..11393
                     /codon_start=1
                     /product="nuclear localization signal of SV40 (simian virus
                     40) large T antigen"
                     /label=SV40 NLS
                     /translation="PKKKRKV"
     CDS             11409..11474
                     /codon_start=1
                     /product="three tandem FLAG(R) epitope tags, followed by an
                     enterokinase cleavage site"
                     /label=3xFLAG
                     /translation="DYKDHDGDYKDHDIDYKDDDDK"
     CDS             11523..11708
                     /codon_start=1
                     /product="Kruppel-associated box (KRAB) transcriptional 
                     repression domain from the human zinc finger protein ZNF10 
                     (Margolin et al., 1994)"
                     /label=KRAB
                     /translation="RTLVTFKDVFVDFTREEWKLLDTAQQIVYRNVMLENYKNLVSLGY
                     QLTKPDVILRLEKGEEP"
     CDS             11718..11771
                     /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             12498..12554
                     /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             12555..12953
                     /codon_start=1
                     /gene="Aspergillus terreus BSD"
                     /product="blasticidin S deaminase"
                     /label=BSD
                     /note="confers resistance to blasticidin"
                     /translation="MAKPLSQEESTLIERATATINSIPISEDYSVASAALSSDGRIFTG
                     VNVYHFTGGPCAELVVLGTAAAAAAGNLTCIVAIGNENRGILSPCGRCRQVLLDLHPGI
                     KAIVKDSDGQPTAVGIRELLPSGYVWEG"
     misc_feature    12978..13566
                     /label=WPRE
                     /note="woodchuck hepatitis virus posttranscriptional 
                     regulatory element"
     primer_bind     complement(13031..13051)
                     /label=WPRE-R
                     /note="WPRE, reverse primer"
     CDS             complement(13449..13460)
                     /codon_start=1
                     /product="Factor Xa recognition and cleavage site"
                     /label=Factor Xa site
                     /translation="IEGR"
     primer_bind     complement(13569..13585)
                     /label=KS primer
                     /note="common sequencing primer, one of multiple similar 
                     variants"
     primer_bind     complement(13570..13586)
                     /label=pBluescriptKS
                     /note="For pBluescript vector"
     LTR             14091..14271
                     /label=5' LTR (truncated)
                     /note="truncated 5' long terminal repeat (LTR) from HIV-1"
     primer_bind     complement(14297..14314)
                     /label=BGH-rev
                     /note="Bovine growth hormone terminator, reverse primer. 
                     Also called BGH reverse"
     polyA_signal    14303..14527
                     /label=bGH poly(A) signal
                     /note="bovine growth hormone polyadenylation signal"
     rep_origin      join(14573..14997,1..4)
                     /direction=RIGHT
                     /label=f1 ori
                     /note="f1 bacteriophage origin of replication; arrow 
                     indicates direction of (+) strand synthesis"
     primer_bind     complement(14660..14679)
                     /label=F1ori-R
                     /note="F1 origin, reverse primer"
     primer_bind     14870..14891
                     /label=F1ori-F
                     /note="F1 origin, forward primer"
ORIGIN
        1 aattaattct gtggaatgtg tgtcagttag ggtgtggaaa gtccccaggc tccccagcag
       61 gcagaagtat gcaaagcatg catctcaatt agtcagcaac caggtgtgga aagtccccag
      121 gctccccagc aggcagaagt atgcaaagca tgcatctcaa ttagtcagca accatagtcc
      181 cgcccctaac tccgcccatc ccgcccctaa ctccgcccag ttccgcccat tctccgcccc
      241 atggctgact aatttttttt atttatgcag aggccgaggc cgcctctgcc tctgagctat
      301 tccagaagta gtgaggaggc ttttttggag gcctaggctt ttgcaaaaag ctcccgggag
      361 cttgtatatc cattttcgga tctgatcagc acgtgttgac aattaatcat cggcatagta
      421 tatcggcata gtataatacg acaaggtgag gaactaaacc atggccaagt tgaccagtgc
      481 cgttccggtg ctcaccgcgc gcgacgtcgc cggagcggtc gagttctgga ccgaccggct
      541 cgggttctcc cgggacttcg tggaggacga cttcgccggt gtggtccggg acgacgtgac
      601 cctgttcatc agcgcggtcc aggaccaggt ggtgccggac aacaccctgg cctgggtgtg
      661 ggtgcgcggc ctggacgagc tgtacgccga gtggtcggag gtcgtgtcca cgaacttccg
      721 ggacgcctcc gggccggcca tgaccgagat cggcgagcag ccgtgggggc gggagttcgc
      781 cctgcgcgac ccggccggca actgcgtgca cttcgtggcc gaggagcagg actgacacgt
      841 gctacgagat ttcgattcca ccgccgcctt ctatgaaagg ttgggcttcg gaatcgtttt
      901 ccgggacgcc ggctggatga tcctccagcg cggggatctc atgctggagt tcttcgccca
      961 ccccaacttg tttattgcag cttataatgg ttacaaataa agcaatagca tcacaaattt
     1021 cacaaataaa gcattttttt cactgcattc tagttgtggt ttgtccaaac tcatcaatgt
     1081 atcttatcat gtctgtatac cgtcgacctc tagctagagc ttggcgtaat catggtcata
     1141 gctgtttcct gtgtgaaatt gttatccgct cacaattcca cacaacatac gagccggaag
     1201 cataaagtgt aaagcctggg gtgcctaatg agtgagctaa ctcacattaa ttgcgttgcg
     1261 ctcactgccc gctttccagt cgggaaacct gtcgtgccag ctgcattaat gaatcggcca
     1321 acgcgcgggg agaggcggtt tgcgtattgg gcgctcttcc gcttcctcgc tcactgactc
     1381 gctgcgctcg gtcgttcggc tgcggcgagc ggtatcagct cactcaaagg cggtaatacg
     1441 gttatccaca gaatcagggg ataacgcagg aaagaacatg tgagcaaaag gccagcaaaa
     1501 ggccaggaac cgtaaaaagg ccgcgttgct ggcgtttttc cataggctcc gcccccctga
     1561 cgagcatcac aaaaatcgac gctcaagtca gaggtggcga aacccgacag gactataaag
     1621 ataccaggcg tttccccctg gaagctccct cgtgcgctct cctgttccga ccctgccgct
     1681 taccggatac ctgtccgcct ttctcccttc gggaagcgtg gcgctttctc atagctcacg
     1741 ctgtaggtat ctcagttcgg tgtaggtcgt tcgctccaag ctgggctgtg tgcacgaacc
     1801 ccccgttcag cccgaccgct gcgccttatc cggtaactat cgtcttgagt ccaacccggt
     1861 aagacacgac ttatcgccac tggcagcagc cactggtaac aggattagca gagcgaggta
     1921 tgtaggcggt gctacagagt tcttgaagtg gtggcctaac tacggctaca ctagaagaac
     1981 agtatttggt atctgcgctc tgctgaagcc agttaccttc ggaaaaagag ttggtagctc
     2041 ttgatccggc aaacaaacca ccgctggtag cggtggtttt tttgtttgca agcagcagat
     2101 tacgcgcaga aaaaaaggat ctcaagaaga tcctttgatc ttttctacgg ggtctgacgc
     2161 tcagtggaac gaaaactcac gttaagggat tttggtcatg agattatcaa aaaggatctt
     2221 cacctagatc cttttaaatt aaaaatgaag ttttaaatca atctaaagta tatatgagta
     2281 aacttggtct gacagttacc aatgcttaat cagtgaggca cctatctcag cgatctgtct
     2341 atttcgttca tccatagttg cctgactccc cgtcgtgtag ataactacga tacgggaggg
     2401 cttaccatct ggccccagtg ctgcaatgat accgcgagac ccacgctcac cggctccaga
     2461 tttatcagca ataaaccagc cagccggaag ggccgagcgc agaagtggtc ctgcaacttt
     2521 atccgcctcc atccagtcta ttaattgttg ccgggaagct agagtaagta gttcgccagt
     2581 taatagtttg cgcaacgttg ttgccattgc tacaggcatc gtggtgtcac gctcgtcgtt
     2641 tggtatggct tcattcagct ccggttccca acgatcaagg cgagttacat gatcccccat
     2701 gttgtgcaaa aaagcggtta gctccttcgg tcctccgatc gttgtcagaa gtaagttggc
     2761 cgcagtgtta tcactcatgg ttatggcagc actgcataat tctcttactg tcatgccatc
     2821 cgtaagatgc ttttctgtga ctggtgagta ctcaaccaag tcattctgag aatagtgtat
     2881 gcggcgaccg agttgctctt gcccggcgtc aatacgggat aataccgcgc cacatagcag
     2941 aactttaaaa gtgctcatca ttggaaaacg ttcttcgggg cgaaaactct caaggatctt
     3001 accgctgttg agatccagtt cgatgtaacc cactcgtgca cccaactgat cttcagcatc
     3061 ttttactttc accagcgttt ctgggtgagc aaaaacagga aggcaaaatg ccgcaaaaaa
     3121 gggaataagg gcgacacgga aatgttgaat actcatactc ttcctttttc aatattattg
     3181 aagcatttat cagggttatt gtctcatgag cggatacata tttgaatgta tttagaaaaa
     3241 taaacaaata ggggttccgc gcacatttcc ccgaaaagtg ccacctgacg tcgacggatc
     3301 gggagatctc ccgatcccct atggtgcact ctcagtacaa tctgctctga tgccgcatag
     3361 ttaagccagt atctgctccc tgcttgtgtg ttggaggtcg ctgagtagtg cgcgagcaaa
     3421 atttaagcta caacaaggca aggcttgacc gacaattgca tgaagaatct gcttagggtt
     3481 aggcgttttg cgctgcttcg cgatgtacgg gccagatata cgcgttgaca ttgattattg
     3541 actagttatt aatagtaatc aattacgggg tcattagttc atagcccata tatggagttc
     3601 cgcgttacat aacttacggt aaatggcccg cctggctgac cgcccaacga cccccgccca
     3661 ttgacgtcaa taatgacgta tgttcccata gtaacgccaa tagggacttt ccattgacgt
     3721 caatgggtgg agtatttacg gtaaactgcc cacttggcag tacatcaagt gtatcatatg
     3781 ccaagtacgc cccctattga cgtcaatgac ggtaaatggc ccgcctggca ttatgcccag
     3841 tacatgacct tatgggactt tcctacttgg cagtacatct acgtattagt catcgctatt
     3901 accatggtga tgcggttttg gcagtacatc aatgggcgtg gatagcggtt tgactcacgg
     3961 ggatttccaa gtctccaccc cattgacgtc aatgggagtt tgttttggca ccaaaatcaa
     4021 cgggactttc caaaatgtcg taacaactcc gccccattga cgcaaatggg cggtaggcgt
     4081 gtacggtggg aggtctatat aagcagcgcg ttttgcctgt actgggtctc tctggttaga
     4141 ccagatctga gcctgggagc tctctggcta actagggaac ccactgctta agcctcaata
     4201 aagcttgcct tgagtgcttc aagtagtgtg tgcccgtctg ttgtgtgact ctggtaacta
     4261 gagatccctc agaccctttt agtcagtgtg gaaaatctct agcagtggcg cccgaacagg
     4321 gacttgaaag cgaaagggaa accagaggag ctctctcgac gcaggactcg gcttgctgaa
     4381 gcgcgcacgg caagaggcga ggggcggcga ctggtgagta cgccaaaaat tttgactagc
     4441 ggaggctaga aggagagaga tgggtgcgag agcgtcagta ttaagcgggg gagaattaga
     4501 tcgcgatggg aaaaaattcg gttaaggcca gggggaaaga aaaaatataa attaaaacat
     4561 atagtatggg caagcaggga gctagaacga ttcgcagtta atcctggcct gttagaaaca
     4621 tcagaaggct gtagacaaat actgggacag ctacaaccat cccttcagac aggatcagaa
     4681 gaacttagat cattatataa tacagtagca accctctatt gtgtgcatca aaggatagag
     4741 ataaaagaca ccaaggaagc tttagacaag atagaggaag agcaaaacaa aagtaagacc
     4801 accgcacagc aagcggccgc tgatcttcag acctggagga ggagatatga gggacaattg
     4861 gagaagtgaa ttatataaat ataaagtagt aaaaattgaa ccattaggag tagcacccac
     4921 caaggcaaag agaagagtgg tgcagagaga aaaaagagca gtgggaatag gagctttgtt
     4981 ccttgggttc ttgggagcag caggaagcac tatgggcgca gcgtcaatga cgctgacggt
     5041 acaggccaga caattattgt ctggtatagt gcagcagcag aacaatttgc tgagggctat
     5101 tgaggcgcaa cagcatctgt tgcaactcac agtctggggc atcaagcagc tccaggcaag
     5161 aatcctggct gtggaaagat acctaaagga tcaacagctc ctggggattt ggggttgctc
     5221 tggaaaactc atttgcacca ctgctgtgcc ttggaatgct agttggagta ataaatctct
     5281 ggaacagatt tggaatcaca cgacctggat ggagtgggac agagaaatta acaattacac
     5341 aagcttaata cactccttaa ttgaagaatc gcaaaaccag caagaaaaga atgaacaaga
     5401 attattggaa ttagataaat gggcaagttt gtggaattgg tttaacataa caaattggct
     5461 gtggtatata aaattattca taatgatagt aggaggcttg gtaggtttaa gaatagtttt
     5521 tgctgtactt tctatagtga atagagttag gcagggatat tcaccattat cgtttcagac
     5581 ccacctccca accccgaggg gacccgacag gcccgaagga atagaagaag aaggtggaga
     5641 gagagacaga gacagatcca ttcgattagt gaacggatcg gcactgcgtg cgccaattct
     5701 gcagacaaat ggcagtattc atccacaatt ttaaaagaaa aggggggatt ggggggtaca
     5761 gtgcagggga aagaatagta gacataatag caacagacat acaaactaaa gaattacaaa
     5821 aacaaattac aaaaattcaa aattttcggg tttattacag ggacagcaga gatccagttt
     5881 ggttaattag ctagctgcaa agatggataa agttttaaac agagaggaat ctttgcagct
     5941 aatggacctt ctaggtcttg aaaggagtgg gaattggctc cggtgcccgt cagtgggcag
     6001 agcgcacatc gcccacagtc cccgagaagt tggggggagg ggtcggcaat tgaaccggtg
     6061 cctagagaag gtggcgcggg gtaaactggg aaagtgatgt cgtgtactgg ctccgccttt
     6121 ttcccgaggg tgggggagaa ccgtatataa gtgcagtagt cgccgtgaac gttctttttc
     6181 gcaacgggtt tgccgccaga acacaggtaa gtgccgtgtg tggttcccgc gggcctggcc
     6241 tctttacggg ttatggccct tgcgtgcctt gaattacttc cacctggctg cagtacgtga
     6301 ttcttgatcc cgagcttcgg gttggaagtg ggtgggagag ttcgaggcct tgcgcttaag
     6361 gagccccttc gcctcgtgct tgagttgagg cctggcctgg gcgctggggc cgccgcgtgc
     6421 gaatctggtg gcaccttcgc gcctgtctcg ctgctttcga taagtctcta gccatttaaa
     6481 atttttgatg acctgctgcg acgctttttt tctggcaaga tagtcttgta aatgcgggcc
     6541 aagatctgca cactggtatt tcggtttttg gggccgcggg cggcgacggg gcccgtgcgt
     6601 cccagcgcac atgttcggcg aggcggggcc tgcgagcgcg gccaccgaga atcggacggg
     6661 ggtagtctca agctggccgg cctgctctgg tgcctggcct cgcgccgccg tgtatcgccc
     6721 cgccctgggc ggcaaggctg gcccggtcgg caccagttgc gtgagcggaa agatggccgc
     6781 ttcccggccc tgctgcaggg agctcaaaat ggaggacgcg gcgctcggga gagcgggcgg
     6841 gtgagtcacc cacacaaagg aaaagggcct ttccgtcctc agccgtcgct tcatgtgact
     6901 ccacggagta ccgggcgccg tccaggcacc tcgattagtt ctcgagcttt tggagtacgt
     6961 cgtctttagg ttggggggag gggttttatg cgatggagtt tccccacact gagtgggtgg
     7021 agactgaagt taggccagct tggcacttga tgtaattctc cttggaattt gccctttttg
     7081 agtttggatc ttggttcatt ctcaagcctc agacagtggt tcaaagtttt tttcttccat
     7141 ttcaggtgtc gtgacgtacg gccaccatga aaaggccggc ggccacgaaa aaggccggcc
     7201 aggcaaaaaa gaaaaaggac aagaagtaca gcatcggcct ggccatcggc accaactctg
     7261 tgggctgggc cgtgatcacc gacgagtaca aggtgcccag caagaaattc aaggtgctgg
     7321 gcaacaccga ccggcacagc atcaagaaga acctgatcgg agccctgctg ttcgacagcg
     7381 gcgaaacagc cgaggccacc cggctgaaga gaaccgccag aagaagatac accagacgga
     7441 agaaccggat ctgctatctg caagagatct tcagcaacga gatggccaag gtggacgaca
     7501 gcttcttcca cagactggaa gagtccttcc tggtggaaga ggataagaag cacgagcggc
     7561 accccatctt cggcaacatc gtggacgagg tggcctacca cgagaagtac cccaccatct
     7621 accacctgag aaagaaactg gtggacagca ccgacaaggc cgacctgcgg ctgatctatc
     7681 tggccctggc ccacatgatc aagttccggg gccacttcct gatcgagggc gacctgaacc
     7741 ccgacaacag cgacgtggac aagctgttca tccagctggt gcagacctac aaccagctgt
     7801 tcgaggaaaa ccccatcaac gccagcggcg tggacgccaa ggccatcctg tctgccagac
     7861 tgagcaagag cagacggctg gaaaatctga tcgcccagct gcccggcgag aagaagaatg
     7921 gcctgttcgg caacctgatt gccctgagcc tgggcctgac ccccaacttc aagagcaact
     7981 tcgacctggc cgaggatgcc aaactgcagc tgagcaagga cacctacgac gacgacctgg
     8041 acaacctgct ggcccagatc ggcgaccagt acgccgacct gtttctggcc gccaagaacc
     8101 tgtccgacgc catcctgctg agcgacatcc tgagagtgaa caccgagatc accaaggccc
     8161 ccctgagcgc ctctatgatc aagagatacg acgagcacca ccaggacctg accctgctga
     8221 aagctctcgt gcggcagcag ctgcctgaga agtacaaaga gattttcttc gaccagagca
     8281 agaacggcta cgccggctac attgacggcg gagccagcca ggaagagttc tacaagttca
     8341 tcaagcccat cctggaaaag atggacggca ccgaggaact gctcgtgaag ctgaacagag
     8401 aggacctgct gcggaagcag cggaccttcg acaacggcag catcccccac cagatccacc
     8461 tgggagagct gcacgccatt ctgcggcggc aggaagattt ttacccattc ctgaaggaca
     8521 accgggaaaa gatcgagaag atcctgacct tccgcatccc ctactacgtg ggccctctgg
     8581 ccaggggaaa cagcagattc gcctggatga ccagaaagag cgaggaaacc atcaccccct
     8641 ggaacttcga ggaagtggtg gacaagggcg cttccgccca gagcttcatc gagcggatga
     8701 ccaacttcga taagaacctg cccaacgaga aggtgctgcc caagcacagc ctgctgtacg
     8761 agtacttcac cgtgtataac gagctgacca aagtgaaata cgtgaccgag ggaatgagaa
     8821 agcccgcctt cctgagcggc gagcagaaaa aggccatcgt ggacctgctg ttcaagacca
     8881 accggaaagt gaccgtgaag cagctgaaag aggactactt caagaaaatc gagtgcttcg
     8941 actccgtgga aatctccggc gtggaagatc ggttcaacgc ctccctgggc acataccacg
     9001 atctgctgaa aattatcaag gacaaggact tcctggacaa tgaggaaaac gaggacattc
     9061 tggaagatat cgtgctgacc ctgacactgt ttgaggacag agagatgatc gaggaacggc
     9121 tgaaaaccta tgcccacctg ttcgacgaca aagtgatgaa gcagctgaag cggcggagat
     9181 acaccggctg gggcaggctg agccggaagc tgatcaacgg catccgggac aagcagtccg
     9241 gcaagacaat cctggatttc ctgaagtccg acggcttcgc caacagaaac ttcatgcagc
     9301 tgatccacga cgacagcctg acctttaaag aggacatcca gaaagcccag gtgtccggcc
     9361 agggcgatag cctgcacgag cacattgcca atctggccgg cagccccgcc attaagaagg
     9421 gcatcctgca gacagtgaag gtggtggacg agctcgtgaa agtgatgggc cggcacaagc
     9481 ccgagaacat cgtgatcgaa atggccagag agaaccagac cacccagaag ggacagaaga
     9541 acagccgcga gagaatgaag cggatcgaag agggcatcaa agagctgggc agccagatcc
     9601 tgaaagaaca ccccgtggaa aacacccagc tgcagaacga gaagctgtac ctgtactacc
     9661 tgcagaatgg gcgggatatg tacgtggacc aggaactgga catcaaccgg ctgtccgact
     9721 acgatgtgga ccacatcgtg cctcagagct ttctgaagga cgactccatc gacaacaagg
     9781 tgctgaccag aagcgacaag gcccggggca agagcgacaa cgtgccctcc gaagaggtcg
     9841 tgaagaagat gaagaactac tggcggcagc tgctgaacgc caagctgatt acccagagaa
     9901 agttcgacaa tctgaccaag gccgagagag gcggcctgag cgaactggat aaggccggct
     9961 tcatcaagag acagctggtg gaaacccggc agatcacaaa gcacgtggca cagatcctgg
    10021 actcccggat gaacactaag tacgacgaga atgacaagct gatccgggaa gtgaaagtga
    10081 tcaccctgaa gtccaagctg gtgtccgatt tccggaagga tttccagttt tacaaagtgc
    10141 gcgagatcaa caactaccac cacgcccacg acgcctacct gaacgccgtc gtgggaaccg
    10201 ccctgatcaa aaagtaccct aagctggaaa gcgagttcgt gtacggcgac tacaaggtgt
    10261 acgacgtgcg gaagatgatc gccaagagcg agcaggaaat cggcaaggct accgccaagt
    10321 acttcttcta cagcaacatc atgaactttt tcaagaccga gattaccctg gccaacggcg
    10381 agatccggaa gcggcctctg atcgagacaa acggcgaaac cggggagatc gtgtgggata
    10441 agggccggga ttttgccacc gtgcggaaag tgctgagcat gccccaagtg aatatcgtga
    10501 aaaagaccga ggtgcagaca ggcggcttca gcaaagagtc tatcctgccc aagaggaaca
    10561 gcgataagct gatcgccaga aagaaggact gggaccctaa gaagtacggc ggcttcgaca
    10621 gccccaccgt ggcctattct gtgctggtgg tggccaaagt ggaaaagggc aagtccaaga
    10681 aactgaagag tgtgaaagag ctgctgggga tcaccatcat ggaaagaagc agcttcgaga
    10741 agaatcccat cgactttctg gaagccaagg gctacaaaga agtgaaaaag gacctgatca
    10801 tcaagctgcc taagtactcc ctgttcgagc tggaaaacgg ccggaagaga atgctggcct
    10861 ctgccggcga actgcagaag ggaaacgaac tggccctgcc ctccaaatat gtgaacttcc
    10921 tgtacctggc cagccactat gagaagctga agggctcccc cgaggataat gagcagaaac
    10981 agctgtttgt ggaacagcac aagcactacc tggacgagat catcgagcag atcagcgagt
    11041 tctccaagag agtgatcctg gccgacgcta atctggacaa agtgctgtcc gcctacaaca
    11101 agcaccggga taagcccatc agagagcagg ccgagaatat catccacctg tttaccctga
    11161 ccaatctggg agcccctgcc gccttcaagt actttgacac caccatcgac cggaagaggt
    11221 acaccagcac caaagaggtg ctggacgcca ccctgatcca ccagagcatc accggcctgt
    11281 acgagacacg gatcgacctg tctcagctgg gaggcgacag cgctggagga ggtggaagcg
    11341 gaggaggagg aagcggagga ggaggtagcg gacctaagaa aaagaggaag gtggcggccg
    11401 ctggatccga ctacaaagac catgacggtg attataaaga tcatgacatc gactacaagg
    11461 atgacgatga caagggtgga tccggatcag gctccgatgc taagtctctg actgcctggt
    11521 ccaggaccct tgtgaccttc aaggacgtct tcgttgattt tacgcgggaa gaatggaaac
    11581 tactagacac agcacaacaa atagtataca gaaacgtaat gctagaaaac tacaaaaacc
    11641 tagtaagcct aggataccaa ctaacaaaac cagacgtaat actaagacta gaaaaaggag
    11701 aagaaccagg atctggagag ggaaggggaa gcctcctaac ttgcggagat gtcgaggaga
    11761 atcctggccc aatggttagt aagggagagg agttgataaa agagaatatg cacatgaaac
    11821 tgtatatgga agggacagtg gacaaccatc acttcaagtg cacctctgag ggagaaggaa
    11881 agccatacga aggcacccag actatgcgaa ttaaggtcgt ggagggaggg cctctcccct
    11941 tcgccttcga tattcttgca acatctttcc tctacgggag caaaaccttc atcaatcaca
    12001 cacaaggcat acctgacttt tttaagcaga gttttccaga gggctttacc tgggagcgtg
    12061 tgactacata cgaagatggg ggtgtgctga cggctacaca ggatacttca cttcaggacg
    12121 gttgtctgat ctacaacgtc aagatcaggg gggtcaattt cacctcgaat ggaccagtaa
    12181 tgcaaaaaaa gaccctggga tgggaagctt ttactgaaac tctctatccc gccgacggcg
    12241 gcctcgaagg cagaaacgat atggctttga agctggttgg cgggtcccat ctaattgcca
    12301 atgccaaaac cacatatagg agcaaaaagc ctgcaaagaa cctgaagatg ccgggtgtgt
    12361 actatgttga ctatagatta gagcggatca aagaggcaaa taacgaaacg tacgtggagc
    12421 agcacgaggt agccgtggcg cgctactgcg atttaccctc caaactgggc cataaactga
    12481 actgtacagg aagcggagct actaacttca gcctgctgaa gcaggctggc gacgtggaag
    12541 aaaacccagg acctatggcc aagcctttgt ctcaagaaga atccaccctc attgaaagag
    12601 caacggctac aatcaacagc atccccatct ctgaagacta cagcgtcgcc agcgcagctc
    12661 tctctagcga cggccgcatc ttcactggtg tcaatgtata tcattttact gggggacctt
    12721 gtgcagaact cgtggtgctg ggcactgctg ctgctgcggc agctggcaac ctgacttgta
    12781 tcgtcgcgat cggaaatgag aacaggggca tcttgagccc ctgcggacgg tgccgacagg
    12841 tgcttctcga tctgcatcct gggatcaaag ccatagtgaa ggacagtgat ggacagccga
    12901 cggcagttgg gattcgtgaa ttgctgccct ctggttatgt gtgggagggc taagaattcg
    12961 atatcaagct tatcggtaat caacctctgg attacaaaat ttgtgaaaga ttgactggta
    13021 ttcttaacta tgttgctcct tttacgctat gtggatacgc tgctttaatg cctttgtatc
    13081 atgctattgc ttcccgtatg gctttcattt tctcctcctt gtataaatcc tggttgctgt
    13141 ctctttatga ggagttgtgg cccgttgtca ggcaacgtgg cgtggtgtgc actgtgtttg
    13201 ctgacgcaac ccccactggt tggggcattg ccaccacctg tcagctcctt tccgggactt
    13261 tcgctttccc cctccctatt gccacggcgg aactcatcgc cgcctgcctt gcccgctgct
    13321 ggacaggggc tcggctgttg ggcactgaca attccgtggt gttgtcgggg aaatcatcgt
    13381 cctttccttg gctgctcgcc tgtgttgcca cctggattct gcgcgggacg tccttctgct
    13441 acgtcccttc ggccctcaat ccagcggacc ttccttcccg cggcctgctg ccggctctgc
    13501 ggcctcttcc gcgtcttcgc cttcgccctc agacgagtcg gatctccctt tgggccgcct
    13561 ccccgcatcg ataccgtcga cctcgagacc tagaaaaaca tggagcaatc acaagtagca
    13621 atacagcagc taccaatgct gattgtgcct ggctagaagc acaagaggag gaggaggtgg
    13681 gttttccagt cacacctcag gtacctttaa gaccaatgac ttacaaggca gctgtagatc
    13741 ttagccactt tttaaaagaa aaggggggac tggaagggct aattcactcc caacgaagac
    13801 aagatatcct tgatctgtgg atctaccaca cacaaggcta cttccctgat tggcagaact
    13861 acacaccagg gccagggatc agatatccac tgacctttgg atggtgctac aagctagtac
    13921 cagttgagca agagaaggta gaagaagcca atgaaggaga gaacacccgc ttgttacacc
    13981 ctgtgagcct gcatgggatg gatgacccgg agagagaagt attagagtgg aggtttgaca
    14041 gccgcctagc atttcatcac atggcccgag agctgcatcc ggactgtact gggtctctct
    14101 ggttagacca gatctgagcc tgggagctct ctggctaact agggaaccca ctgcttaagc
    14161 ctcaataaag cttgccttga gtgcttcaag tagtgtgtgc ccgtctgttg tgtgactctg
    14221 gtaactagag atccctcaga cccttttagt cagtgtggaa aatctctagc agggcccgtt
    14281 taaacccgct gatcagcctc gactgtgcct tctagttgcc agccatctgt tgtttgcccc
    14341 tcccccgtgc cttccttgac cctggaaggt gccactccca ctgtcctttc ctaataaaat
    14401 gaggaaattg catcgcattg tctgagtagg tgtcattcta ttctgggggg tggggtgggg
    14461 caggacagca agggggagga ttgggaagac aatagcaggc atgctgggga tgcggtgggc
    14521 tctatggctt ctgaggcgga aagaaccagc tggggctcta gggggtatcc ccacgcgccc
    14581 tgtagcggcg cattaagcgc ggcgggtgtg gtggttacgc gcagcgtgac cgctacactt
    14641 gccagcgccc tagcgcccgc tcctttcgct ttcttccctt cctttctcgc cacgttcgcc
    14701 ggctttcccc gtcaagctct aaatcggggg ctccctttag ggttccgatt tagtgcttta
    14761 cggcacctcg accccaaaaa acttgattag ggtgatggtt cacgtagtgg gccatcgccc
    14821 tgatagacgg tttttcgccc tttgacgttg gagtccacgt tctttaatag tggactcttg
    14881 ttccaaactg gaacaacact caaccctatc tcggtctatt cttttgattt ataagggatt
    14941 ttgccgattt cggcctattg gttaaaaaat gagctgattt aacaaaaatt taacgcg
//
