pcDNA3.1-CibN-dCas9-CibN

LOCUS       pcDNA3.1-CibN-dCas9-CibN               10635 bp ds-DNA     circular SYN 12-MAY-2021
DEFINITION  Expresses CibN-dCas9-CibN in mammalian cells.
ACCESSION   .
VERSION     .
KEYWORDS    pcDNA3.1-CibN-dCas9-CibN
SOURCE      synthetic DNA construct
  ORGANISM  synthetic DNA construct
REFERENCE   1  (bases 1 to 10635)
  AUTHORS   Polstein LR, Gersbach CA
  TITLE     A light-inducible CRISPR-Cas9 system for control of endogenous gene 
            activation.
  JOURNAL   Nat Chem Biol. 2015 Feb 9. doi: 10.1038/nchembio.1753.
  PUBMED    25664691
REFERENCE   2  (bases 1 to 10635)
  AUTHORS   .
  TITLE     Direct Submission
  JOURNAL   Exported May 12, 2021 from SnapGene Server 1.1.58
            http://www.biofeng.com
FEATURES             Location/Qualifiers
     source          1..10635
                     /organism="synthetic DNA construct"
                     /mol_type="other DNA"
     primer_bind     complement(66..85)
                     /label=F1ori-R
                     /note="F1 origin, reverse primer"
     primer_bind     276..297
                     /label=F1ori-F
                     /note="F1 origin, forward primer"
     primer_bind     complement(416..436)
                     /label=pBABE 3'
                     /note="SV40 enhancer, reverse primer for pBABE vectors"
     promoter        421..750
                     /label=SV40 promoter
                     /note="SV40 enhancer and early promoter"
     rep_origin      601..736
                     /label=SV40 ori
                     /note="SV40 origin of replication"
     primer_bind     663..682
                     /label=SV40pro-F
                     /note="SV40 promoter/origin, forward primer"
     CDS             817..1611
                     /codon_start=1
                     /gene="aph(3')-II (or nptII)"
                     /product="aminoglycoside phosphotransferase from Tn5"
                     /label=NeoR/KanR
                     /note="confers resistance to neomycin, kanamycin, and G418 
                     (Geneticin(R))"
                     /translation="MIEQDGLHAGSPAAWVERLFGYDWAQQTIGCSDAAVFRLSAQGRP
                     VLFVKTDLSGALNELQDEAARLSWLATTGVPCAAVLDVVTEAGRDWLLLGEVPGQDLLS
                     SHLAPAEKVSIMADAMRRLHTLDPATCPFDHQAKHRIERARTRMEAGLVDQDDLDEEHQ
                     GLAPAELFARLKARMPDGEDLVVTHGDACLPNIMVENGRFSGFIDCGRLGVADRYQDIA
                     LATRDIAEELGGEWADRFLVLYGIAAPDSQRIAFYRLLDEFF"
     primer_bind     complement(871..890)
                     /label=Neo-R
                     /note="Neomycin resistance gene, reverse primer"
     primer_bind     1481..1500
                     /label=Neo-F
                     /note="Neomycin resistance gene, forward primer"
     polyA_signal    1785..1906
                     /label=SV40 poly(A) signal
                     /note="SV40 polyadenylation signal"
     primer_bind     complement(1822..1841)
                     /label=SV40pA-R
                     /note="SV40 polyA, reverse primer"
     primer_bind     1876..1895
                     /label=EBV-rev
                     /note="SV40 polyA terminator, reverse primer"
     primer_bind     complement(1955..1971)
                     /label=M13 rev
                     /note="common sequencing primer, one of multiple similar 
                     variants"
     primer_bind     complement(1955..1971)
                     /label=M13 Reverse
                     /note="In lacZ gene. Also called M13-rev"
     primer_bind     complement(1968..1990)
                     /label=M13/pUC Reverse
                     /note="In lacZ gene"
     protein_bind    1979..1995
                     /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(2003..2033)
                     /label=lac promoter
                     /note="promoter for the E. coli lac operon"
     protein_bind    2048..2069
                     /label=CAP binding site
                     /bound_moiety="E. coli catabolite activator protein"
                     /note="CAP binding activates transcription in the presence 
                     of cAMP."
     primer_bind     complement(2186..2203)
                     /label=L4440
                     /note="L4440 vector, forward primer"
     rep_origin      complement(2357..2945)
                     /direction=LEFT
                     /label=ori
                     /note="high-copy-number ColE1/pMB1/pBR322/pUC origin of 
                     replication"
     primer_bind     complement(2437..2456)
                     /label=pBR322ori-F
                     /note="pBR322 origin, forward primer"
     CDS             complement(3116..3976)
                     /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     3739..3758
                     /label=Amp-R
                     /note="Ampicillin resistance gene, reverse primer"
     promoter        complement(3977..4081)
                     /gene="bla"
                     /label=AmpR promoter
     primer_bind     complement(4156..4175)
                     /label=pRS-marker
                     /note="pRS vectors, use to sequence yeast selectable 
                     marker"
     enhancer        4347..4726
                     /label=CMV enhancer
                     /note="human cytomegalovirus immediate early enhancer"
     promoter        4727..4930
                     /label=CMV promoter
                     /note="human cytomegalovirus (CMV) immediate early 
                     promoter"
     primer_bind     4881..4901
                     /label=CMV-F
                     /note="Human CMV immediate early promoter, forward primer"
     primer_bind     4975..4994
                     /label=T7
                     /note="T7 promoter, forward primer"
     promoter        4975..4993
                     /label=T7 promoter
                     /note="promoter for bacteriophage T7 RNA polymerase"
     CDS             5067..5573
                     /codon_start=1
                     /product="N-terminal portion of the CIB1 transcription 
                     factor from Arabidopsis thaliana"
                     /label=CIBN
                     /note="binds to CRY2 (cryptochrome 2) that has absorbed 
                     blue light (Kennedy et al., 2010)"
                     /translation="NGAIGGDLLLNFPDMSVLERQRAHLKYLNPTFDSPLAGFFADSSM
                     ITGGEMDSYLSTAGLNLPMMYGETTVEGDSRLSISPETTLGTGNFKKRKFDTETKDCNE
                     KKKKMTMNRDDLVEEGEEEKSKITEQNNGSTKSIKKMKHKAKKEENNFSNDSSKVTKEL
                     EKTDYI"
     CDS             5580..5603
                     /codon_start=1
                     /product="FLAG(R) epitope tag, followed by an enterokinase 
                     cleavage site"
                     /label=FLAG
                     /translation="DYKDDDDK"
     CDS             5610..5630
                     /codon_start=1
                     /product="nuclear localization signal of SV40 (simian virus
                     40) large T antigen"
                     /label=SV40 NLS
                     /translation="PKKKRKV"
     CDS             5640..9743
                     /codon_start=1
                     /product="catalytically dead mutant of the Cas9 
                     endonuclease from the Streptococcus pyogenes Type II 
                     CRISPR/Cas system"
                     /label=dCas9
                     /note="RNA-guided DNA-binding protein that lacks 
                     endonuclease activity due to the D10A mutation in the RuvC 
                     catalytic domain and the H840A mutation in the HNH 
                     catalytic domain"
                     /translation="MDKKYSIGLAIGTNSVGWAVITDEYKVPSKKFKVLGNTDRHSIKK
                     NLIGALLFDSGETAEATRLKRTARRRYTRRKNRICYLQEIFSNEMAKVDDSFFHRLEES
                     FLVEEDKKHERHPIFGNIVDEVAYHEKYPTIYHLRKKLVDSTDKADLRLIYLALAHMIK
                     FRGHFLIEGDLNPDNSDVDKLFIQLVQTYNQLFEENPINASGVDAKAILSARLSKSRRL
                     ENLIAQLPGEKKNGLFGNLIALSLGLTPNFKSNFDLAEDAKLQLSKDTYDDDLDNLLAQ
                     IGDQYADLFLAAKNLSDAILLSDILRVNTEITKAPLSASMIKRYDEHHQDLTLLKALVR
                     QQLPEKYKEIFFDQSKNGYAGYIDGGASQEEFYKFIKPILEKMDGTEELLVKLNREDLL
                     RKQRTFDNGSIPHQIHLGELHAILRRQEDFYPFLKDNREKIEKILTFRIPYYVGPLARG
                     NSRFAWMTRKSEETITPWNFEEVVDKGASAQSFIERMTNFDKNLPNEKVLPKHSLLYEY
                     FTVYNELTKVKYVTEGMRKPAFLSGEQKKAIVDLLFKTNRKVTVKQLKEDYFKKIECFD
                     SVEISGVEDRFNASLGTYHDLLKIIKDKDFLDNEENEDILEDIVLTLTLFEDREMIEER
                     LKTYAHLFDDKVMKQLKRRRYTGWGRLSRKLINGIRDKQSGKTILDFLKSDGFANRNFM
                     QLIHDDSLTFKEDIQKAQVSGQGDSLHEHIANLAGSPAIKKGILQTVKVVDELVKVMGR
                     HKPENIVIEMARENQTTQKGQKNSRERMKRIEEGIKELGSQILKEHPVENTQLQNEKLY
                     LYYLQNGRDMYVDQELDINRLSDYDVDAIVPQSFLKDDSIDNKVLTRSDKNRGKSDNVP
                     SEEVVKKMKNYWRQLLNAKLITQRKFDNLTKAERGGLSELDKAGFIKRQLVETRQITKH
                     VAQILDSRMNTKYDENDKLIREVKVITLKSKLVSDFRKDFQFYKVREINNYHHAHDAYL
                     NAVVGTALIKKYPKLESEFVYGDYKVYDVRKMIAKSEQEIGKATAKYFFYSNIMNFFKT
                     EITLANGEIRKRPLIETNGETGEIVWDKGRDFATVRKVLSMPQVNIVKKTEVQTGGFSK
                     ESILPKRNSDKLIARKKDWDPKKYGGFDSPTVAYSVLVVAKVEKGKSKKLKSVKELLGI
                     TIMERSSFEKNPIDFLEAKGYKEVKKDLIIKLPKYSLFELENGRKRMLASAGELQKGNE
                     LALPSKYVNFLYLASHYEKLKGSPEDNEQKQLFVEQHKHYLDEIIEQISEFSKRVILAD
                     ANLDKVLSAYNKHRDKPIREQAENIIHLFTLTNLGAPAAFKYFDTTIDRKRYTSTKEVL
                     DATLIHQSITGLYETRIDLSQLGGD"
     CDS             9768..9788
                     /codon_start=1
                     /product="nuclear localization signal of SV40 (simian virus
                     40) large T antigen"
                     /label=SV40 NLS
                     /translation="PKKKRKV"
     CDS             9801..10310
                     /codon_start=1
                     /product="N-terminal portion of the CIB1 transcription 
                     factor from Arabidopsis thaliana"
                     /label=CIBN
                     /note="binds to CRY2 (cryptochrome 2) that has absorbed 
                     blue light (Kennedy et al., 2010)"
                     /translation="MNGAIGGDLLLNFPDMSVLERQRAHLKYLNPTFDSPLAGFFADSS
                     MITGGEMDSYLSTAGLNLPMMYGETTVEGDSRLSISPETTLGTGNFKKRKFDTETKDCN
                     EKKKKMTMNRDDLVEEGEEEKSKITEQNNGSTKSIKKMKHKAKKEENNFSNDSSKVTKE
                     LEKTDYI"
     primer_bind     complement(10338..10355)
                     /label=BGH-rev
                     /note="Bovine growth hormone terminator, reverse primer. 
                     Also called BGH reverse"
     polyA_signal    10344..10568
                     /label=bGH poly(A) signal
                     /note="bovine growth hormone polyadenylation signal"
     rep_origin      10614..407
                     /direction=RIGHT
                     /label=f1 ori
                     /note="f1 bacteriophage origin of replication; arrow 
                     indicates direction of (+) strand synthesis"
ORIGIN
        1 aagcgcggcg ggtgtggtgg ttacgcgcag cgtgaccgct acacttgcca gcgccctagc
       61 gcccgctcct ttcgctttct tcccttcctt tctcgccacg ttcgccggct ttccccgtca
      121 agctctaaat cgggggctcc ctttagggtt ccgatttagt gctttacggc acctcgaccc
      181 caaaaaactt gattagggtg atggttcacg tagtgggcca tcgccctgat agacggtttt
      241 tcgccctttg acgttggagt ccacgttctt taatagtgga ctcttgttcc aaactggaac
      301 aacactcaac cctatctcgg tctattcttt tgatttataa gggattttgc cgatttcggc
      361 ctattggtta aaaaatgagc tgatttaaca aaaatttaac gcgaattaat tctgtggaat
      421 gtgtgtcagt tagggtgtgg aaagtcccca ggctccccag caggcagaag tatgcaaagc
      481 atgcatctca attagtcagc aaccaggtgt ggaaagtccc caggctcccc agcaggcaga
      541 agtatgcaaa gcatgcatct caattagtca gcaaccatag tcccgcccct aactccgccc
      601 atcccgcccc taactccgcc cagttccgcc cattctccgc cccatggctg actaattttt
      661 tttatttatg cagaggccga ggccgcctct gcctctgagc tattccagaa gtagtgagga
      721 ggcttttttg gaggcctagg cttttgcaaa aagctcccgg gagcttgtat atccattttc
      781 ggatctgatc aagagacagg atgaggatcg tttcgcatga ttgaacaaga tggattgcac
      841 gcaggttctc cggccgcttg ggtggagagg ctattcggct atgactgggc acaacagaca
      901 atcggctgct ctgatgccgc cgtgttccgg ctgtcagcgc aggggcgccc ggttcttttt
      961 gtcaagaccg acctgtccgg tgccctgaat gaactgcagg acgaggcagc gcggctatcg
     1021 tggctggcca cgacgggcgt tccttgcgca gctgtgctcg acgttgtcac tgaagcggga
     1081 agggactggc tgctattggg cgaagtgccg gggcaggatc tcctgtcatc tcaccttgct
     1141 cctgccgaga aagtatccat catggctgat gcaatgcggc ggctgcatac gcttgatccg
     1201 gctacctgcc cattcgacca ccaagcgaaa catcgcatcg agcgagcacg tactcggatg
     1261 gaagccggtc ttgtcgatca ggatgatctg gacgaagagc atcaggggct cgcgccagcc
     1321 gaactgttcg ccaggctcaa ggcgcgcatg cccgacggcg aggatctcgt cgtgacccat
     1381 ggcgatgcct gcttgccgaa tatcatggtg gaaaatggcc gcttttctgg attcatcgac
     1441 tgtggccggc tgggtgtggc ggaccgctat caggacatag cgttggctac ccgtgatatt
     1501 gctgaagagc ttggcggcga atgggctgac cgcttcctcg tgctttacgg tatcgccgct
     1561 cccgattcgc agcgcatcgc cttctatcgc cttcttgacg agttcttctg agcgggactc
     1621 tggggttcga aatgaccgac caagcgacgc ccaacctgcc atcacgagat ttcgattcca
     1681 ccgccgcctt ctatgaaagg ttgggcttcg gaatcgtttt ccgggacgcc ggctggatga
     1741 tcctccagcg cggggatctc atgctggagt tcttcgccca ccccaacttg tttattgcag
     1801 cttataatgg ttacaaataa agcaatagca tcacaaattt cacaaataaa gcattttttt
     1861 cactgcattc tagttgtggt ttgtccaaac tcatcaatgt atcttatcat gtctgtatac
     1921 cgtcgacctc tagctagagc ttggcgtaat catggtcata gctgtttcct gtgtgaaatt
     1981 gttatccgct cacaattcca cacaacatac gagccggaag cataaagtgt aaagcctggg
     2041 gtgcctaatg agtgagctaa ctcacattaa ttgcgttgcg ctcactgccc gctttccagt
     2101 cgggaaacct gtcgtgccag ctgcattaat gaatcggcca acgcgcgggg agaggcggtt
     2161 tgcgtattgg gcgctcttcc gcttcctcgc tcactgactc gctgcgctcg gtcgttcggc
     2221 tgcggcgagc ggtatcagct cactcaaagg cggtaatacg gttatccaca gaatcagggg
     2281 ataacgcagg aaagaacatg tgagcaaaag gccagcaaaa ggccaggaac cgtaaaaagg
     2341 ccgcgttgct ggcgtttttc cataggctcc gcccccctga cgagcatcac aaaaatcgac
     2401 gctcaagtca gaggtggcga aacccgacag gactataaag ataccaggcg tttccccctg
     2461 gaagctccct cgtgcgctct cctgttccga ccctgccgct taccggatac ctgtccgcct
     2521 ttctcccttc gggaagcgtg gcgctttctc atagctcacg ctgtaggtat ctcagttcgg
     2581 tgtaggtcgt tcgctccaag ctgggctgtg tgcacgaacc ccccgttcag cccgaccgct
     2641 gcgccttatc cggtaactat cgtcttgagt ccaacccggt aagacacgac ttatcgccac
     2701 tggcagcagc cactggtaac aggattagca gagcgaggta tgtaggcggt gctacagagt
     2761 tcttgaagtg gtggcctaac tacggctaca ctagaagaac agtatttggt atctgcgctc
     2821 tgctgaagcc agttaccttc ggaaaaagag ttggtagctc ttgatccggc aaacaaacca
     2881 ccgctggtag cggtggtttt tttgtttgca agcagcagat tacgcgcaga aaaaaaggat
     2941 ctcaagaaga tcctttgatc ttttctacgg ggtctgacgc tcagtggaac gaaaactcac
     3001 gttaagggat tttggtcatg agattatcaa aaaggatctt cacctagatc cttttaaatt
     3061 aaaaatgaag ttttaaatca atctaaagta tatatgagta aacttggtct gacagttacc
     3121 aatgcttaat cagtgaggca cctatctcag cgatctgtct atttcgttca tccatagttg
     3181 cctgactccc cgtcgtgtag ataactacga tacgggaggg cttaccatct ggccccagtg
     3241 ctgcaatgat accgcgagac ccacgctcac cggctccaga tttatcagca ataaaccagc
     3301 cagccggaag ggccgagcgc agaagtggtc ctgcaacttt atccgcctcc atccagtcta
     3361 ttaattgttg ccgggaagct agagtaagta gttcgccagt taatagtttg cgcaacgttg
     3421 ttgccattgc tacaggcatc gtggtgtcac gctcgtcgtt tggtatggct tcattcagct
     3481 ccggttccca acgatcaagg cgagttacat gatcccccat gttgtgcaaa aaagcggtta
     3541 gctccttcgg tcctccgatc gttgtcagaa gtaagttggc cgcagtgtta tcactcatgg
     3601 ttatggcagc actgcataat tctcttactg tcatgccatc cgtaagatgc ttttctgtga
     3661 ctggtgagta ctcaaccaag tcattctgag aatagtgtat gcggcgaccg agttgctctt
     3721 gcccggcgtc aatacgggat aataccgcgc cacatagcag aactttaaaa gtgctcatca
     3781 ttggaaaacg ttcttcgggg cgaaaactct caaggatctt accgctgttg agatccagtt
     3841 cgatgtaacc cactcgtgca cccaactgat cttcagcatc ttttactttc accagcgttt
     3901 ctgggtgagc aaaaacagga aggcaaaatg ccgcaaaaaa gggaataagg gcgacacgga
     3961 aatgttgaat actcatactc ttcctttttc aatattattg aagcatttat cagggttatt
     4021 gtctcatgag cggatacata tttgaatgta tttagaaaaa taaacaaata ggggttccgc
     4081 gcacatttcc ccgaaaagtg ccacctgacg tcgacggatc gggagatctc ccgatcccct
     4141 atggtgcact ctcagtacaa tctgctctga tgccgcatag ttaagccagt atctgctccc
     4201 tgcttgtgtg ttggaggtcg ctgagtagtg cgcgagcaaa atttaagcta caacaaggca
     4261 aggcttgacc gacaattgca tgaagaatct gcttagggtt aggcgttttg cgctgcttcg
     4321 cgatgtacgg gccagatata cgcgttgaca ttgattattg actagttatt aatagtaatc
     4381 aattacgggg tcattagttc atagcccata tatggagttc cgcgttacat aacttacggt
     4441 aaatggcccg cctggctgac cgcccaacga cccccgccca ttgacgtcaa taatgacgta
     4501 tgttcccata gtaacgccaa tagggacttt ccattgacgt caatgggtgg agtatttacg
     4561 gtaaactgcc cacttggcag tacatcaagt gtatcatatg ccaagtacgc cccctattga
     4621 cgtcaatgac ggtaaatggc ccgcctggca ttatgcccag tacatgacct tatgggactt
     4681 tcctacttgg cagtacatct acgtattagt catcgctatt accatggtga tgcggttttg
     4741 gcagtacatc aatgggcgtg gatagcggtt tgactcacgg ggatttccaa gtctccaccc
     4801 cattgacgtc aatgggagtt tgttttggca ccaaaatcaa cgggactttc caaaatgtcg
     4861 taacaactcc gccccattga cgcaaatggg cggtaggcgt gtacggtggg aggtctatat
     4921 aagcagagct ctctggctaa ctagagaacc cactgcttac tggcttatcg aaattaatac
     4981 gactcactat agggagaccc aagctggcta gcgccatgga ctacaaagac catgacggtg
     5041 attataaaga tcatgacatc gatataaatg gagctatagg aggtgacctt ttgctcaatt
     5101 ttcctgacat gtcggtccta gagcgccaaa gggctcacct caagtacctc aatcccacct
     5161 ttgattctcc tctcgccggc ttctttgccg attcttcaat gattaccggc ggcgagatgg
     5221 acagctatct ttcgactgcc ggtttgaatc ttccgatgat gtacggtgag acgacggtgg
     5281 aaggtgattc aagactctca atttcgccgg aaacgacgct tgggactgga aatttcaaga
     5341 aacggaagtt tgatacagag actaaggatt gtaatgagaa gaagaagaag atgacgatga
     5401 acagagatga cctagtagaa gaaggagaag aagagaagtc gaaaataaca gagcaaaaca
     5461 atgggagcac aaaaagcatc aagaagatga aacacaaagc caagaaagaa gagaacaatt
     5521 tctctaatga ttcatctaaa gtgacgaagg aattggagaa aacggattat attcatcacg
     5581 attacaagga tgacgatgac aagatggccc ccaagaagaa gaggaaggtg ggccgcggaa
     5641 tggataagaa atactcaata ggcttagcta tcggcacaaa tagcgtcgga tgggcggtga
     5701 tcactgatga atataaggtt ccgtctaaaa agttcaaggt tctgggaaat acagaccgcc
     5761 acagtatcaa aaaaaatctt ataggggctc ttttatttga cagtggagag acagcggaag
     5821 cgactcgtct caaacggaca gctcgtagaa ggtatacacg tcggaagaat cgtatttgtt
     5881 atctacagga gattttttca aatgagatgg cgaaagtaga tgatagtttc tttcatcgac
     5941 ttgaagagtc ttttttggtg gaagaagaca agaagcatga acgtcatcct atttttggaa
     6001 atatagtaga tgaagttgct tatcatgaga aatatccaac tatctatcat ctgcgaaaaa
     6061 aattggtaga ttctactgat aaagcggatt tgcgcttaat ctatttggcc ttagcgcata
     6121 tgattaagtt tcgtggtcat tttttgattg agggagattt aaatcctgat aatagtgatg
     6181 tggacaaact atttatccag ttggtacaaa cctacaatca attatttgaa gaaaacccta
     6241 ttaacgcaag tggagtagat gctaaagcga ttctttctgc acgattgagt aaatcaagac
     6301 gattagaaaa tctcattgct cagctccccg gtgagaagaa aaatggctta tttgggaatc
     6361 tcattgcttt gtcattgggt ttgaccccta attttaaatc aaattttgat ttggcagaag
     6421 atgctaaatt acagctttca aaagatactt acgatgatga tttagataat ttattggcgc
     6481 aaattggaga tcaatatgct gatttgtttt tggcagctaa gaatttatca gatgctattt
     6541 tactttcaga tatcctaaga gtaaatactg aaataactaa ggctccccta tcagcttcaa
     6601 tgattaaacg ctacgatgaa catcatcaag acttgactct tttaaaagct ttagttcgac
     6661 aacaacttcc agaaaagtat aaagaaatct tttttgatca atcaaaaaac ggatatgcag
     6721 gttatattga tgggggagct agccaagaag aattttataa atttatcaaa ccaattttag
     6781 aaaaaatgga tggtactgag gaattattgg tgaaactaaa tcgtgaagat ttgctgcgca
     6841 agcaacggac ctttgacaac ggctctattc cccatcaaat tcacttgggt gagctgcatg
     6901 ctattttgag aagacaagaa gacttttatc catttttaaa agacaatcgt gagaagattg
     6961 aaaaaatctt gacttttcga attccttatt atgttggtcc attggcgcgt ggcaatagtc
     7021 gttttgcatg gatgactcgg aagtctgaag aaacaattac cccatggaat tttgaagaag
     7081 ttgtcgataa aggtgcttca gctcaatcat ttattgaacg catgacaaac tttgataaaa
     7141 atcttccaaa tgaaaaagta ctaccaaaac atagtttgct ttatgagtat tttacggttt
     7201 ataacgaatt gacaaaggtc aaatatgtta ctgaaggaat gcgaaaacca gcatttcttt
     7261 caggtgaaca gaagaaagcc attgttgatt tactcttcaa aacaaatcga aaagtaaccg
     7321 ttaagcaatt aaaagaagat tatttcaaaa aaatagaatg ttttgatagt gttgaaattt
     7381 caggagttga agatagattt aatgcttcat taggtaccta ccatgatttg ctaaaaatta
     7441 ttaaagataa agattttttg gataatgaag aaaatgaaga tatcttagag gatattgttt
     7501 taacattgac cttatttgaa gatagggaga tgattgagga aagacttaaa acatatgctc
     7561 acctctttga tgataaggtg atgaaacagc ttaaacgtcg ccgttatact ggttggggac
     7621 gtttgtctcg aaaattgatt aatggtatta gggataagca atctggcaaa acaatattag
     7681 attttttgaa atcagatggt tttgccaatc gcaattttat gcagctgatc catgatgata
     7741 gtttgacatt taaagaagac attcaaaaag cacaagtgtc tggacaaggc gatagtttac
     7801 atgaacatat tgcaaattta gctggtagcc ctgctattaa aaaaggtatt ttacagactg
     7861 taaaagttgt tgatgaattg gtcaaagtaa tggggcggca taagccagaa aatatcgtta
     7921 ttgaaatggc acgtgaaaat cagacaactc aaaagggcca gaaaaattcg cgagagcgta
     7981 tgaaacgaat cgaagaaggt atcaaagaat taggaagtca gattcttaaa gagcatcctg
     8041 ttgaaaatac tcaattgcaa aatgaaaagc tctatctcta ttatctccaa aatggaagag
     8101 acatgtatgt ggaccaagaa ttagatatta atcgtttaag tgattatgat gtcgatgcca
     8161 ttgttccaca aagtttcctt aaagacgatt caatagacaa taaggtctta acgcgttctg
     8221 ataaaaatcg tggtaaatcg gataacgttc caagtgaaga agtagtcaaa aagatgaaaa
     8281 actattggag acaacttcta aacgccaagt taatcactca acgtaagttt gataatttaa
     8341 cgaaagctga acgtggaggt ttgagtgaac ttgataaagc tggttttatc aaacgccaat
     8401 tggttgaaac tcgccaaatc actaagcatg tggcacaaat tttggatagt cgcatgaata
     8461 ctaaatacga tgaaaatgat aaacttattc gagaggttaa agtgattacc ttaaaatcta
     8521 aattagtttc tgacttccga aaagatttcc aattctataa agtacgtgag attaacaatt
     8581 accatcatgc ccatgatgcg tatctaaatg ccgtcgttgg aactgctttg attaagaaat
     8641 atccaaaact tgaatcggag tttgtctatg gtgattataa agtttatgat gttcgtaaaa
     8701 tgattgctaa gtctgagcaa gaaataggca aagcaaccgc aaaatatttc ttttactcta
     8761 atatcatgaa cttcttcaaa acagaaatta cacttgcaaa tggagagatt cgcaaacgcc
     8821 ctctaatcga aactaatggg gaaactggag aaattgtctg ggataaaggg cgagattttg
     8881 ccacagtgcg caaagtattg tccatgcccc aagtcaatat tgtcaagaaa acagaagtac
     8941 agacaggcgg attctccaag gagtcaattt taccaaaaag aaattcggac aagcttattg
     9001 ctcgtaaaaa agactgggat ccaaaaaaat atggtggttt tgatagtcca acggtagctt
     9061 attcagtcct agtggttgct aaggtggaaa aagggaaatc gaagaagtta aaatccgtta
     9121 aagagttact agggatcaca attatggaaa gaagttcctt tgaaaaaaat ccgattgact
     9181 ttttagaagc taaaggatat aaggaagtta aaaaagactt aatcattaaa ctacctaaat
     9241 atagtctttt tgagttagaa aacggtcgta aacggatgct ggctagtgcc ggagaattac
     9301 aaaaaggaaa tgagctggct ctgccaagca aatatgtgaa ttttttatat ttagctagtc
     9361 attatgaaaa gttgaagggt agtccagaag ataacgaaca aaaacaattg tttgtggagc
     9421 agcataagca ttatttagat gagattattg agcaaatcag tgaattttct aagcgtgtta
     9481 ttttagcaga tgccaattta gataaagttc ttagtgcata taacaaacat agagacaaac
     9541 caatacgtga acaagcagaa aatattattc atttatttac gttgacgaat cttggagctc
     9601 ccgctgcttt taaatatttt gatacaacaa ttgatcgtaa acgatatacg tctacaaaag
     9661 aagttttaga tgccactctt atccatcaat ccatcactgg tctttatgaa acacgcattg
     9721 atttgagtca gctaggaggt gacccaattg ccggatccaa ggctagcccg aaaaagaaac
     9781 gcaaagttgg gcgcgccacc atgaatggag ctataggagg tgaccttttg ctcaattttc
     9841 ctgacatgtc ggtcctagag cgccaaaggg ctcacctcaa gtacctcaat cccacctttg
     9901 attctcctct cgccggcttc tttgccgatt cttcaatgat taccggcggc gagatggaca
     9961 gctatctttc gactgccggt ttgaatcttc cgatgatgta cggtgagacg acggtggaag
    10021 gtgattcaag actctcaatt tcgccggaaa cgacgcttgg gactggaaat ttcaagaaac
    10081 ggaagtttga tacagagact aaggattgta atgagaagaa gaagaagatg acgatgaaca
    10141 gagatgacct agtagaagaa ggagaagaag agaagtcgaa aataacagag caaaacaatg
    10201 ggagcacaaa aagcatcaag aagatgaaac acaaagccaa gaaagaagag aacaatttct
    10261 ctaatgattc atctaaagtg acgaaggaat tggagaaaac ggattatatt taaacttaag
    10321 tttaaaccgc tgatcagcct cgactgtgcc ttctagttgc cagccatctg ttgtttgccc
    10381 ctcccccgtg ccttccttga ccctggaagg tgccactccc actgtccttt cctaataaaa
    10441 tgaggaaatt gcatcgcatt gtctgagtag gtgtcattct attctggggg gtggggtggg
    10501 gcaggacagc aagggggagg attgggaaga caatagcagg catgctgggg atgcggtggg
    10561 ctctatggct tctgaggcgg aaagaaccag ctggggctct agggggtatc cccacgcgcc
    10621 ctgtagcggc gcatt
//