SpdCas9 VPR

LOCUS       Exported               14489 bp ds-DNA     circular SYN 28-SEP-2022
DEFINITION  Expresses FLAG tagged SpdCas9 VPR.
ACCESSION   .
VERSION     .
KEYWORDS    SpdCas9 VPR
SOURCE      synthetic DNA construct
  ORGANISM  synthetic DNA construct
REFERENCE   1  (bases 1 to 14489)
  AUTHORS   Escobar M, Li J, Patel A, Liu S, Xu Q, Hilton IB
  TITLE     Quantification of Genome Editing and Transcriptional Control 
            Capabilities Reveals Hierarchies among Diverse CRISPR/Cas Systems in
            Human Cells
  JOURNAL   ACS Synth. Biol. 2022
REFERENCE   2  (bases 1 to 14489)
  AUTHORS   .
  TITLE     Direct Submission
  JOURNAL   Exported Sep 28, 2022 from SnapGene Server 1.1.58
            http://www.snapgene.com
FEATURES             Location/Qualifiers
     source          1..14489
                     /organism="synthetic DNA construct"
                     /mol_type="other DNA"
     promoter        1..199
                     /label=CMV promoter
                     /note="human cytomegalovirus (CMV) immediate early 
                     promoter"
     primer_bind     154..174
                     /label=CMV-F
                     /note="Human CMV immediate early promoter, forward primer"
     LTR             217..397
                     /label=5' LTR (truncated)
                     /note="truncated 5' long terminal repeat (LTR) from HIV-1"
     misc_feature    444..569
                     /label=HIV-1 Psi
                     /note="packaging signal of human immunodeficiency virus 
                     type 1"
     misc_feature    1062..1295
                     /label=RRE
                     /note="The Rev response element (RRE) of HIV-1 allows for 
                     Rev-dependent mRNA export from the nucleus to the 
                     cytoplasm."
     CDS             1480..1524
                     /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    1822..1939
                     /label=cPPT/CTS
                     /note="central polypurine tract and central termination 
                     sequence of HIV-1"
     misc_RNA        2071..2146
                     /label=gRNA scaffold
                     /note="guide RNA scaffold for the Streptococcus pyogenes 
                     CRISPR/Cas9 system"
     promoter        2208..2419
                     /label=EF-1-alpha core promoter
                     /note="core promoter for human elongation factor 
                     EF-1-alpha"
     regulatory      2438..2447
                     /regulatory_class="other"
                     /note="vertebrate consensus sequence for strong initiation 
                     of translation (Kozak, 1987)"
     CDS             2444..6547
                     /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             6548..6595
                     /codon_start=1
                     /product="bipartite nuclear localization signal from 
                     nucleoplasmin"
                     /label=nucleoplasmin NLS
                     /translation="KRPAATKKAGQAKKKK"
     CDS             6596..6619
                     /codon_start=1
                     /product="FLAG(R) epitope tag, followed by an enterokinase 
                     cleavage site"
                     /label=FLAG
                     /translation="DYKDDDDK"
     protein_bind    6662..6686
                     /gene="mutant version of attB"
                     /label=attB1
                     /bound_moiety="BP Clonase(TM)"
                     /note="recombination site for the Gateway(R) BP reaction"
     CDS             6716..6865
                     /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             6890..6910
                     /codon_start=1
                     /product="nuclear localization signal of SV40 (simian virus
                     40) large T antigen"
                     /label=SV40 NLS
                     /translation="PKKKRKV"
     CDS             7340..7696
                     /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             7715..8284
                     /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             8294..8350
                     /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             8351..8947
                     /codon_start=1
                     /gene="pac from Streptomyces alboniger"
                     /product="puromycin N-acetyltransferase"
                     /label=PuroR
                     /note="confers resistance to puromycin"
                     /translation="TEYKPTVRLATRDDVPRAVRTLAAAFADYPATRHTVDPDRHIERV
                     TELQELFLTRVGLDIGKVWVADDGAAVAVWTTPESVEAGAVFAEIGPRMAELSGSRLAA
                     QQQMEGLLAPHRPKEPAWFLATVGVSPDHQGKGLGSAVVLPGVEAAERAGVPAFLETSA
                     PRNLPFYERLGFTVTADVEVPEGPRTWCMTRKPGA"
     primer_bind     8844..8864
                     /label=Puro-F
                     /note="Puromycin resistance gene, forward primer"
     misc_feature    8963..9551
                     /label=WPRE
                     /note="woodchuck hepatitis virus posttranscriptional 
                     regulatory element"
     primer_bind     complement(9016..9036)
                     /label=WPRE-R
                     /note="WPRE, reverse primer"
     CDS             complement(9434..9445)
                     /codon_start=1
                     /product="Factor Xa recognition and cleavage site"
                     /label=Factor Xa site
                     /translation="IEGR"
     LTR             9623..9856
                     /label=3' LTR (Delta-U3)
                     /note="self-inactivating 3' long terminal repeat (LTR) from
                     HIV-1"
     primer_bind     complement(9882..9899)
                     /label=BGH-rev
                     /note="Bovine growth hormone terminator, reverse primer. 
                     Also called BGH reverse"
     polyA_signal    9888..10112
                     /label=bGH poly(A) signal
                     /note="bovine growth hormone polyadenylation signal"
     rep_origin      10158..10586
                     /direction=RIGHT
                     /label=f1 ori
                     /note="f1 bacteriophage origin of replication; arrow 
                     indicates direction of (+) strand synthesis"
     primer_bind     complement(10245..10264)
                     /label=F1ori-R
                     /note="F1 origin, reverse primer"
     primer_bind     10455..10476
                     /label=F1ori-F
                     /note="F1 origin, forward primer"
     primer_bind     complement(10595..10615)
                     /label=pBABE 3'
                     /note="SV40 enhancer, reverse primer for pBABE vectors"
     promoter        10600..10929
                     /label=SV40 promoter
                     /note="SV40 enhancer and early promoter"
     rep_origin      10780..10915
                     /label=SV40 ori
                     /note="SV40 origin of replication"
     primer_bind     10842..10861
                     /label=SV40pro-F
                     /note="SV40 promoter/origin, forward primer"
     promoter        10977..11024
                     /label=EM7 promoter
                     /note="synthetic bacterial promoter "
     CDS             11043..11417
                     /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    11547..11668
                     /label=SV40 poly(A) signal
                     /note="SV40 polyadenylation signal"
     primer_bind     complement(11584..11603)
                     /label=SV40pA-R
                     /note="SV40 polyA, reverse primer"
     primer_bind     11638..11657
                     /label=EBV-rev
                     /note="SV40 polyA terminator, reverse primer"
     primer_bind     complement(11717..11733)
                     /label=M13 rev
                     /note="common sequencing primer, one of multiple similar 
                     variants"
     primer_bind     complement(11717..11733)
                     /label=M13 Reverse
                     /note="In lacZ gene. Also called M13-rev"
     primer_bind     complement(11730..11752)
                     /label=M13/pUC Reverse
                     /note="In lacZ gene"
     protein_bind    11741..11757
                     /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(11765..11795)
                     /label=lac promoter
                     /note="promoter for the E. coli lac operon"
     protein_bind    11810..11831
                     /label=CAP binding site
                     /bound_moiety="E. coli catabolite activator protein"
                     /note="CAP binding activates transcription in the presence 
                     of cAMP."
     primer_bind     complement(11948..11965)
                     /label=L4440
                     /note="L4440 vector, forward primer"
     rep_origin      complement(12119..12707)
                     /direction=LEFT
                     /label=ori
                     /note="high-copy-number ColE1/pMB1/pBR322/pUC origin of 
                     replication"
     primer_bind     complement(12199..12218)
                     /label=pBR322ori-F
                     /note="pBR322 origin, forward primer"
     CDS             complement(12878..13738)
                     /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     13501..13520
                     /label=Amp-R
                     /note="Ampicillin resistance gene, reverse primer"
     promoter        complement(13739..13843)
                     /gene="bla"
                     /label=AmpR promoter
     primer_bind     complement(13918..13937)
                     /label=pRS-marker
                     /note="pRS vectors, use to sequence yeast selectable 
                     marker"
     enhancer        14109..14488
                     /label=CMV enhancer
                     /note="human cytomegalovirus immediate early enhancer"
ORIGIN
        1 tgatgcggtt ttggcagtac atcaatgggc gtggatagcg gtttgactca cggggatttc
       61 caagtctcca ccccattgac gtcaatggga gtttgttttg gcaccaaaat caacgggact
      121 ttccaaaatg tcgtaacaac tccgccccat tgacgcaaat gggcggtagg cgtgtacggt
      181 gggaggtcta tataagcagc gcgttttgcc tgtactgggt ctctctggtt agaccagatc
      241 tgagcctggg agctctctgg ctaactaggg aacccactgc ttaagcctca ataaagcttg
      301 ccttgagtgc ttcaagtagt gtgtgcccgt ctgttgtgtg actctggtaa ctagagatcc
      361 ctcagaccct tttagtcagt gtggaaaatc tctagcagtg gcgcccgaac agggacttga
      421 aagcgaaagg gaaaccagag gagctctctc gacgcaggac tcggcttgct gaagcgcgca
      481 cggcaagagg cgaggggcgg cgactggtga gtacgccaaa aattttgact agcggaggct
      541 agaaggagag agatgggtgc gagagcgtca gtattaagcg ggggagaatt agatcgcgat
      601 gggaaaaaat tcggttaagg ccagggggaa agaaaaaata taaattaaaa catatagtat
      661 gggcaagcag ggagctagaa cgattcgcag ttaatcctgg cctgttagaa acatcagaag
      721 gctgtagaca aatactggga cagctacaac catcccttca gacaggatca gaagaactta
      781 gatcattata taatacagta gcaaccctct attgtgtgca tcaaaggata gagataaaag
      841 acaccaagga agctttagac aagatagagg aagagcaaaa caaaagtaag accaccgcac
      901 agcaagcggc cgctgatctt cagacctgga ggaggagata tgagggacaa ttggagaagt
      961 gaattatata aatataaagt agtaaaaatt gaaccattag gagtagcacc caccaaggca
     1021 aagagaagag tggtgcagag agaaaaaaga gcagtgggaa taggagcttt gttccttggg
     1081 ttcttgggag cagcaggaag cactatgggc gcagcgtcaa tgacgctgac ggtacaggcc
     1141 agacaattat tgtctggtat agtgcagcag cagaacaatt tgctgagggc tattgaggcg
     1201 caacagcatc tgttgcaact cacagtctgg ggcatcaagc agctccaggc aagaatcctg
     1261 gctgtggaaa gatacctaaa ggatcaacag ctcctgggga tttggggttg ctctggaaaa
     1321 ctcatttgca ccactgctgt gccttggaat gctagttgga gtaataaatc tctggaacag
     1381 atttggaatc acacgacctg gatggagtgg gacagagaaa ttaacaatta cacaagctta
     1441 atacactcct taattgaaga atcgcaaaac cagcaagaaa agaatgaaca agaattattg
     1501 gaattagata aatgggcaag tttgtggaat tggtttaaca taacaaattg gctgtggtat
     1561 ataaaattat tcataatgat agtaggaggc ttggtaggtt taagaatagt ttttgctgta
     1621 ctttctatag tgaatagagt taggcaggga tattcaccat tatcgtttca gacccacctc
     1681 ccaaccccga ggggacccga caggcccgaa ggaatagaag aagaaggtgg agagagagac
     1741 agagacagat ccattcgatt agtgaacgga tcggcactgc gtgcgccaat tctgcagaca
     1801 aatggcagta ttcatccaca attttaaaag aaaagggggg attggggggt acagtgcagg
     1861 ggaaagaata gtagacataa tagcaacaga catacaaact aaagaattac aaaaacaaat
     1921 tacaaaaatt caaaattttc gggtttatta cagggacagc agagatccag tttggttaat
     1981 taaggtaccg atatcactag tgttccatta actgcgctaa aaataatttt taaatctttt
     2041 ttagcttctt gctctttttt gtacgtctct gttttagagc tagaaatagc aagttaaaat
     2101 aaggctagtc cgttatcaac ttgaaaaagt ggcaccgagt cggtgctttt ttgaattcgc
     2161 tagctaggtc ttgaaaggag tgggaattgg ctccggtgcc cgtcagtggg cagagcgcac
     2221 atcgcccaca gtccccgaga agttgggggg aggggtcggc aattgatccg gtgcctagag
     2281 aaggtggcgc ggggtaaact gggaaagtga tgtcgtgtac tggctccgcc tttttcccga
     2341 gggtggggga gaaccgtata taagtgcagt agtcgccgtg aacgttcttt ttcgcaacgg
     2401 gtttgccgcc agaacacagg accggttcta gagcgctgcc accatggaca agaagtacag
     2461 catcggcctg gccatcggca ccaactctgt gggctgggcc gtgatcaccg acgagtacaa
     2521 ggtgcccagc aagaaattca aggtgctggg caacaccgac cggcacagca tcaagaagaa
     2581 cctgatcgga gccctgctgt tcgacagcgg cgaaacagcc gaggccaccc ggctgaagag
     2641 aaccgccaga agaagataca ccagacggaa gaaccggatc tgctatctgc aagagatctt
     2701 cagcaacgag atggccaagg tggacgacag cttcttccac agactggaag agtccttcct
     2761 ggtggaagag gataagaagc acgagcggca ccccatcttc ggcaacatcg tggacgaggt
     2821 ggcctaccac gagaagtacc ccaccatcta ccacctgaga aagaaactgg tggacagcac
     2881 cgacaaggcc gacctgcggc tgatctatct ggccctggcc cacatgatca agttccgggg
     2941 ccacttcctg atcgagggcg acctgaaccc cgacaacagc gacgtggaca agctgttcat
     3001 ccagctggtg cagacctaca accagctgtt cgaggaaaac cccatcaacg ccagcggcgt
     3061 ggacgccaag gccatcctgt ctgccagact gagcaagagc agacggctgg aaaatctgat
     3121 cgcccagctg cccggcgaga agaagaatgg cctgttcgga aacctgattg ccctgagcct
     3181 gggcctgacc cccaacttca agagcaactt cgacctggcc gaggatgcca aactgcagct
     3241 gagcaaggac acctacgacg acgacctgga caacctgctg gcccagatcg gcgaccagta
     3301 cgccgacctg tttctggccg ccaagaacct gtccgacgcc atcctgctga gcgacatcct
     3361 gagagtgaac accgagatca ccaaggcccc cctgagcgcc tctatgatca agagatacga
     3421 cgagcaccac caggacctga ccctgctgaa agctctcgtg cggcagcagc tgcctgagaa
     3481 gtacaaagag attttcttcg accagagcaa gaacggctac gccggctaca ttgacggcgg
     3541 agccagccag gaagagttct acaagttcat caagcccatc ctggaaaaga tggacggcac
     3601 cgaggaactg ctcgtgaagc tgaacagaga ggacctgctg cggaagcagc ggaccttcga
     3661 caacggcagc atcccccacc agatccacct gggagagctg cacgccattc tgcggcggca
     3721 ggaagatttt tacccattcc tgaaggacaa ccgggaaaag atcgagaaga tcctgacctt
     3781 ccgcatcccc tactacgtgg gccctctggc caggggaaac agcagattcg cctggatgac
     3841 cagaaagagc gaggaaacca tcaccccctg gaacttcgag gaagtggtgg acaagggcgc
     3901 ttccgcccag agcttcatcg agcggatgac caacttcgat aagaacctgc ccaacgagaa
     3961 ggtgctgccc aagcacagcc tgctgtacga gtacttcacc gtgtataacg agctgaccaa
     4021 agtgaaatac gtgaccgagg gaatgagaaa gcccgccttc ctgagcggcg agcagaaaaa
     4081 ggccatcgtg gacctgctgt tcaagaccaa ccggaaagtg accgtgaagc agctgaaaga
     4141 ggactacttc aagaaaatcg agtgcttcga ctccgtggaa atctccggcg tggaagatcg
     4201 gttcaacgcc tccctgggca cataccacga tctgctgaaa attatcaagg acaaggactt
     4261 cctggacaat gaggaaaacg aggacattct ggaagatatc gtgctgaccc tgacactgtt
     4321 tgaggacaga gagatgatcg aggaacggct gaaaacctat gcccacctgt tcgacgacaa
     4381 agtgatgaag cagctgaagc ggcggagata caccggctgg ggcaggctga gccggaagct
     4441 gatcaacggc atccgggaca agcagtccgg caagacaatc ctggatttcc tgaagtccga
     4501 cggcttcgcc aacagaaact tcatgcagct gatccacgac gacagcctga cctttaaaga
     4561 ggacatccag aaagcccagg tgtccggcca gggcgatagc ctgcacgagc acattgccaa
     4621 tctggccggc agccccgcca ttaagaaggg catcctgcag acagtgaagg tggtggacga
     4681 gctcgtgaaa gtgatgggcc ggcacaagcc cgagaacatc gtgatcgaaa tggccagaga
     4741 gaaccagacc acccagaagg gacagaagaa cagccgcgag agaatgaagc ggatcgaaga
     4801 gggcatcaaa gagctgggca gccagatcct gaaagaacac cccgtggaaa acacccagct
     4861 gcagaacgag aagctgtacc tgtactacct gcagaatggg cgggatatgt acgtggacca
     4921 ggaactggac atcaaccggc tgtccgacta cgatgtggac gccatcgtgc ctcagagctt
     4981 tctgaaggac gactccatcg acaacaaggt gctgaccaga agcgacaaga accggggcaa
     5041 gagcgacaac gtgccctccg aagaggtcgt gaagaagatg aagaactact ggcggcagct
     5101 gctgaacgcc aagctgatta cccagagaaa gttcgacaat ctgaccaagg ccgagagagg
     5161 cggcctgagc gaactggata aggccggctt catcaagaga cagctggtgg aaacccggca
     5221 gatcacaaag cacgtggcac agatcctgga ctcccggatg aacactaagt acgacgagaa
     5281 tgacaagctg atccgggaag tgaaagtgat caccctgaag tccaagctgg tgtccgattt
     5341 ccggaaggat ttccagtttt acaaagtgcg cgagatcaac aactaccacc acgcccacga
     5401 cgcctacctg aacgccgtcg tgggaaccgc cctgatcaaa aagtacccta agctggaaag
     5461 cgagttcgtg tacggcgact acaaggtgta cgacgtgcgg aagatgatcg ccaagagcga
     5521 gcaggaaatc ggcaaggcta ccgccaagta cttcttctac agcaacatca tgaacttttt
     5581 caagaccgag attaccctgg ccaacggcga gatccggaag cggcctctga tcgagacaaa
     5641 cggcgaaacc ggggagatcg tgtgggataa gggccgggat tttgccaccg tgcggaaagt
     5701 gctgagcatg ccccaagtga atatcgtgaa aaagaccgag gtgcagacag gcggcttcag
     5761 caaagagtct atcctgccca agaggaacag cgataagctg atcgccagaa agaaggactg
     5821 ggaccctaag aagtacggcg gcttcgacag ccccaccgtg gcctattctg tgctggtggt
     5881 ggccaaagtg gaaaagggca agtccaagaa actgaagagt gtgaaagagc tgctggggat
     5941 caccatcatg gaaagaagca gcttcgagaa gaatcccatc gactttctgg aagccaaggg
     6001 ctacaaagaa gtgaaaaagg acctgatcat caagctgcct aagtactccc tgttcgagct
     6061 ggaaaacggc cggaagagaa tgctggcctc tgccggcgaa ctgcagaagg gaaacgaact
     6121 ggccctgccc tccaaatatg tgaacttcct gtacctggcc agccactatg agaagctgaa
     6181 gggctccccc gaggataatg agcagaaaca gctgtttgtg gaacagcaca agcactacct
     6241 ggacgagatc atcgagcaga tcagcgagtt ctccaagaga gtgatcctgg ccgacgctaa
     6301 tctggacaaa gtgctgtccg cctacaacaa gcaccgggat aagcccatca gagagcaggc
     6361 cgagaatatc atccacctgt ttaccctgac caatctggga gcccctgccg ccttcaagta
     6421 ctttgacacc accatcgacc ggaagaggta caccagcacc aaagaggtgc tggacgccac
     6481 cctgatccac cagagcatca ccggcctgta cgagacacgg atcgacctgt ctcagctggg
     6541 aggcgacaag cgacctgccg ccacaaagaa ggctggacag gctaagaaga agaaagatta
     6601 caaagacgat gacgataagg gatcctcgcc agggatccgt cgacttgacg cgttgatatc
     6661 aacaagtttg tacaaaaaag caggctacaa agaggccagc ggttccggac gggctgacgc
     6721 attggacgat tttgatctgg atatgctggg aagtgacgcc ctcgatgatt ttgaccttga
     6781 catgcttggt tcggatgccc ttgatgactt tgacctcgac atgctcggca gtgacgccct
     6841 tgatgatttc gacctggaca tgctgattaa ctctagaagt tccggatctc cgaaaaagaa
     6901 acgcaaagtt ggtagccagt acctgcccga caccgacgac cggcaccgga tcgaggaaaa
     6961 gcggaagcgg acctacgaga cattcaagag catcatgaag aagtccccct tcagcggccc
     7021 caccgaccct agacctccac ctagaagaat cgccgtgccc agcagatcca gcgccagcgt
     7081 gccaaaacct gccccccagc cttacccctt caccagcagc ctgagcacca tcaactacga
     7141 cgagttccct accatggtgt tccccagcgg ccagatctct caggcctctg ctctggctcc
     7201 agcccctcct caggtgctgc ctcaggctcc tgctcctgca ccagctccag ccatggtgtc
     7261 tgcactggct caggcaccag cacccgtgcc tgtgctggct cctggacctc cacaggctgt
     7321 ggctccacca gcccctaaac ctacacaggc cggcgagggc acactgtctg aagctctgct
     7381 gcagctgcag ttcgacgacg aggatctggg agccctgctg ggaaacagca ccgatcctgc
     7441 cgtgttcacc gacctggcca gcgtggacaa cagcgagttc cagcagctgc tgaaccaggg
     7501 catccctgtg gcccctcaca ccaccgagcc catgctgatg gaataccccg aggccatcac
     7561 ccggctcgtg acaggcgctc agaggcctcc tgatccagct cctgcccctc tgggagcacc
     7621 aggcctgcct aatggactgc tgtctggcga cgaggacttc agctctatcg ccgatatgga
     7681 tttctcagcc ttgctgggct ctggcagcgg cagccgggat tccagggaag ggatgttttt
     7741 gccgaagcct gaggccggct ccgctattag tgacgtgttt gagggccgcg aggtgtgcca
     7801 gccaaaacga atccggccat ttcatcctcc aggaagtcca tgggccaacc gcccactccc
     7861 cgccagcctc gcaccaacac caaccggtcc agtacatgag ccagtcgggt cactgacccc
     7921 ggcaccagtc cctcagccac tggatccagc gcccgcagtg actcccgagg ccagtcacct
     7981 gttggaggat cccgatgaag agacgagcca ggctgtcaaa gcccttcggg agatggccga
     8041 tactgtgatt ccccagaagg aagaggctgc aatctgtggc caaatggacc tttcccatcc
     8101 gcccccaagg ggccatctgg atgagctgac aaccacactt gagtccatga ccgaggatct
     8161 gaacctggac tcacccctga ccccggaatt gaacgagatt ctggatacct tcctgaacga
     8221 cgagtgcctc ttgcatgcca tgcatatcag cacaggactg tccatcttcg acacatctct
     8281 gtttggatcc ggcgcaacaa acttctctct gctgaaacaa gccggagatg tcgaagagaa
     8341 tcctggaccg accgagtaca agcccacggt gcgcctcgcc acccgcgacg acgtccccag
     8401 ggccgtacgc accctcgccg ccgcgttcgc cgactacccc gccacgcgcc acaccgtcga
     8461 tccggaccgc cacatcgagc gggtcaccga gctgcaagaa ctcttcctca cgcgcgtcgg
     8521 gctcgacatc ggcaaggtgt gggtcgcgga cgacggcgcc gcggtggcgg tctggaccac
     8581 gccggagagc gtcgaagcgg gggcggtgtt cgccgagatc ggcccgcgca tggccgagtt
     8641 gagcggttcc cggctggccg cgcagcaaca gatggaaggc ctcctggcgc cgcaccggcc
     8701 caaggagccc gcgtggttcc tggccaccgt cggagtctcg cccgaccacc agggcaaggg
     8761 tctgggcagc gccgtcgtgc tccccggagt ggaggcggcc gagcgcgccg gggtgcccgc
     8821 cttcctggag acctccgcgc cccgcaacct ccccttctac gagcggctcg gcttcaccgt
     8881 caccgccgac gtcgaggtgc ccgaaggacc gcgcacctgg tgcatgaccc gcaagcccgg
     8941 tgcctgaacg cgttaagtcg acaatcaacc tctggattac aaaatttgtg aaagattgac
     9001 tggtattctt aactatgttg ctccttttac gctatgtgga tacgctgctt taatgccttt
     9061 gtatcatgct attgcttccc gtatggcttt cattttctcc tccttgtata aatcctggtt
     9121 gctgtctctt tatgaggagt tgtggcccgt tgtcaggcaa cgtggcgtgg tgtgcactgt
     9181 gtttgctgac gcaaccccca ctggttgggg cattgccacc acctgtcagc tcctttccgg
     9241 gactttcgct ttccccctcc ctattgccac ggcggaactc atcgccgcct gccttgcccg
     9301 ctgctggaca ggggctcggc tgttgggcac tgacaattcc gtggtgttgt cggggaaatc
     9361 atcgtccttt ccttggctgc tcgcctgtgt tgccacctgg attctgcgcg ggacgtcctt
     9421 ctgctacgtc ccttcggccc tcaatccagc ggaccttcct tcccgcggcc tgctgccggc
     9481 tctgcggcct cttccgcgtc ttcgccttcg ccctcagacg agtcggatct ccctttgggc
     9541 cgcctccccg cgtcgacttt aagaccaatg acttacaagg cagctgtaga tcttagccac
     9601 tttttaaaag aaaagggggg actggaaggg ctaattcact cccaacgaag acaagatctg
     9661 ctttttgctt gtactgggtc tctctggtta gaccagatct gagcctggga gctctctggc
     9721 taactaggga acccactgct taagcctcaa taaagcttgc cttgagtgct tcaagtagtg
     9781 tgtgcccgtc tgttgtgtga ctctggtaac tagagatccc tcagaccctt ttagtcagtg
     9841 tggaaaatct ctagcagggc ccgtttaaac ccgctgatca gcctcgactg tgccttctag
     9901 ttgccagcca tctgttgttt gcccctcccc cgtgccttcc ttgaccctgg aaggtgccac
     9961 tcccactgtc ctttcctaat aaaatgagga aattgcatcg cattgtctga gtaggtgtca
    10021 ttctattctg gggggtgggg tggggcagga cagcaagggg gaggattggg aagacaatag
    10081 caggcatgct ggggatgcgg tgggctctat ggcttctgag gcggaaagaa ccagctgggg
    10141 ctctaggggg tatccccacg cgccctgtag cggcgcatta agcgcggcgg gtgtggtggt
    10201 tacgcgcagc gtgaccgcta cacttgccag cgccctagcg cccgctcctt tcgctttctt
    10261 cccttccttt ctcgccacgt tcgccggctt tccccgtcaa gctctaaatc gggggctccc
    10321 tttagggttc cgatttagtg ctttacggca cctcgacccc aaaaaacttg attagggtga
    10381 tggttcacgt agtgggccat cgccctgata gacggttttt cgccctttga cgttggagtc
    10441 cacgttcttt aatagtggac tcttgttcca aactggaaca acactcaacc ctatctcggt
    10501 ctattctttt gatttataag ggattttgcc gatttcggcc tattggttaa aaaatgagct
    10561 gatttaacaa aaatttaacg cgaattaatt ctgtggaatg tgtgtcagtt agggtgtgga
    10621 aagtccccag gctccccagc aggcagaagt atgcaaagca tgcatctcaa ttagtcagca
    10681 accaggtgtg gaaagtcccc aggctcccca gcaggcagaa gtatgcaaag catgcatctc
    10741 aattagtcag caaccatagt cccgccccta actccgccca tcccgcccct aactccgccc
    10801 agttccgccc attctccgcc ccatggctga ctaatttttt ttatttatgc agaggccgag
    10861 gccgcctctg cctctgagct attccagaag tagtgaggag gcttttttgg aggcctaggc
    10921 ttttgcaaaa agctcccggg agcttgtata tccattttcg gatctgatca gcacgtgttg
    10981 acaattaatc atcggcatag tatatcggca tagtataata cgacaaggtg aggaactaaa
    11041 ccatggccaa gttgaccagt gccgttccgg tgctcaccgc gcgcgacgtc gccggagcgg
    11101 tcgagttctg gaccgaccgg ctcgggttct cccgggactt cgtggaggac gacttcgccg
    11161 gtgtggtccg ggacgacgtg accctgttca tcagcgcggt ccaggaccag gtggtgccgg
    11221 acaacaccct ggcctgggtg tgggtgcgcg gcctggacga gctgtacgcc gagtggtcgg
    11281 aggtcgtgtc cacgaacttc cgggacgcct ccgggccggc catgaccgag atcggcgagc
    11341 agccgtgggg gcgggagttc gccctgcgcg acccggccgg caactgcgtg cacttcgtgg
    11401 ccgaggagca ggactgacac gtgctacgag atttcgattc caccgccgcc ttctatgaaa
    11461 ggttgggctt cggaatcgtt ttccgggacg ccggctggat gatcctccag cgcggggatc
    11521 tcatgctgga gttcttcgcc caccccaact tgtttattgc agcttataat ggttacaaat
    11581 aaagcaatag catcacaaat ttcacaaata aagcattttt ttcactgcat tctagttgtg
    11641 gtttgtccaa actcatcaat gtatcttatc atgtctgtat accgtcgacc tctagctaga
    11701 gcttggcgta atcatggtca tagctgtttc ctgtgtgaaa ttgttatccg ctcacaattc
    11761 cacacaacat acgagccgga agcataaagt gtaaagcctg gggtgcctaa tgagtgagct
    11821 aactcacatt aattgcgttg cgctcactgc ccgctttcca gtcgggaaac ctgtcgtgcc
    11881 agctgcatta atgaatcggc caacgcgcgg ggagaggcgg tttgcgtatt gggcgctctt
    11941 ccgcttcctc gctcactgac tcgctgcgct cggtcgttcg gctgcggcga gcggtatcag
    12001 ctcactcaaa ggcggtaata cggttatcca cagaatcagg ggataacgca ggaaagaaca
    12061 tgtgagcaaa aggccagcaa aaggccagga accgtaaaaa ggccgcgttg ctggcgtttt
    12121 tccataggct ccgcccccct gacgagcatc acaaaaatcg acgctcaagt cagaggtggc
    12181 gaaacccgac aggactataa agataccagg cgtttccccc tggaagctcc ctcgtgcgct
    12241 ctcctgttcc gaccctgccg cttaccggat acctgtccgc ctttctccct tcgggaagcg
    12301 tggcgctttc tcatagctca cgctgtaggt atctcagttc ggtgtaggtc gttcgctcca
    12361 agctgggctg tgtgcacgaa ccccccgttc agcccgaccg ctgcgcctta tccggtaact
    12421 atcgtcttga gtccaacccg gtaagacacg acttatcgcc actggcagca gccactggta
    12481 acaggattag cagagcgagg tatgtaggcg gtgctacaga gttcttgaag tggtggccta
    12541 actacggcta cactagaaga acagtatttg gtatctgcgc tctgctgaag ccagttacct
    12601 tcggaaaaag agttggtagc tcttgatccg gcaaacaaac caccgctggt agcggtggtt
    12661 tttttgtttg caagcagcag attacgcgca gaaaaaaagg atctcaagaa gatcctttga
    12721 tcttttctac ggggtctgac gctcagtgga acgaaaactc acgttaaggg attttggtca
    12781 tgagattatc aaaaaggatc ttcacctaga tccttttaaa ttaaaaatga agttttaaat
    12841 caatctaaag tatatatgag taaacttggt ctgacagtta ccaatgctta atcagtgagg
    12901 cacctatctc agcgatctgt ctatttcgtt catccatagt tgcctgactc cccgtcgtgt
    12961 agataactac gatacgggag ggcttaccat ctggccccag tgctgcaatg ataccgcgag
    13021 acccacgctc accggctcca gatttatcag caataaacca gccagccgga agggccgagc
    13081 gcagaagtgg tcctgcaact ttatccgcct ccatccagtc tattaattgt tgccgggaag
    13141 ctagagtaag tagttcgcca gttaatagtt tgcgcaacgt tgttgccatt gctacaggca
    13201 tcgtggtgtc acgctcgtcg tttggtatgg cttcattcag ctccggttcc caacgatcaa
    13261 ggcgagttac atgatccccc atgttgtgca aaaaagcggt tagctccttc ggtcctccga
    13321 tcgttgtcag aagtaagttg gccgcagtgt tatcactcat ggttatggca gcactgcata
    13381 attctcttac tgtcatgcca tccgtaagat gcttttctgt gactggtgag tactcaacca
    13441 agtcattctg agaatagtgt atgcggcgac cgagttgctc ttgcccggcg tcaatacggg
    13501 ataataccgc gccacatagc agaactttaa aagtgctcat cattggaaaa cgttcttcgg
    13561 ggcgaaaact ctcaaggatc ttaccgctgt tgagatccag ttcgatgtaa cccactcgtg
    13621 cacccaactg atcttcagca tcttttactt tcaccagcgt ttctgggtga gcaaaaacag
    13681 gaaggcaaaa tgccgcaaaa aagggaataa gggcgacacg gaaatgttga atactcatac
    13741 tcttcctttt tcaatattat tgaagcattt atcagggtta ttgtctcatg agcggataca
    13801 tatttgaatg tatttagaaa aataaacaaa taggggttcc gcgcacattt ccccgaaaag
    13861 tgccacctga cgtcgacgga tcgggagatc tcccgatccc ctatggtgca ctctcagtac
    13921 aatctgctct gatgccgcat agttaagcca gtatctgctc cctgcttgtg tgttggaggt
    13981 cgctgagtag tgcgcgagca aaatttaagc tacaacaagg caaggcttga ccgacaattg
    14041 catgaagaat ctgcttaggg ttaggcgttt tgcgctgctt cgcgatgtac gggccagata
    14101 tacgcgttga cattgattat tgactagtta ttaatagtaa tcaattacgg ggtcattagt
    14161 tcatagccca tatatggagt tccgcgttac ataacttacg gtaaatggcc cgcctggctg
    14221 accgcccaac gacccccgcc cattgacgtc aataatgacg tatgttccca tagtaacgcc
    14281 aatagggact ttccattgac gtcaatgggt ggagtattta cggtaaactg cccacttggc
    14341 agtacatcaa gtgtatcata tgccaagtac gccccctatt gacgtcaatg acggtaaatg
    14401 gcccgcctgg cattatgccc agtacatgac cttatgggac tttcctactt ggcagtacat
    14461 ctacgtatta gtcatcgcta ttaccatgg
//