pLV hU6-gRNA(anti-sense) hUbC-VP64-dCas9-VP64-T2A-GFP

LOCUS       pLV hU6-gRNA(anti-sense) hUbC-VP64-dCas9-VP64-T2A-GFP               14993 bp ds-DNA     circular SYN 12-MAY-2021
DEFINITION  expresses a single gRNA and VP64-dCas9-VP64.
ACCESSION   .
VERSION     .
KEYWORDS    pLV hU6-gRNA(anti-sense) hUbC-VP64-dCas9-VP64-T2A-GFP
SOURCE      synthetic DNA construct
  ORGANISM  synthetic DNA construct
REFERENCE   1  (bases 1 to 14993)
  AUTHORS   Gersbach C
  TITLE     Multiplex CRISPR/Cas9 Genome Engineering for Directing Myogenic 
            Cellular Differentiation in Primary Human Cells
  JOURNAL   Unpublished
REFERENCE   2  (bases 1 to 14993)
  AUTHORS   .
  TITLE     Direct Submission
  JOURNAL   Exported May 12, 2021 from SnapGene Server 1.1.58
            http://www.snapgene.com
FEATURES             Location/Qualifiers
     source          1..14993
                     /organism="synthetic DNA construct"
                     /mol_type="other DNA"
     enhancer        1..380
                     /label=CMV enhancer
                     /note="human cytomegalovirus immediate early enhancer"
     promoter        382..580
                     /label=CMV promoter
                     /note="human cytomegalovirus (CMV) immediate early 
                     promoter"
     primer_bind     535..555
                     /label=CMV-F
                     /note="Human CMV immediate early promoter, forward primer"
     LTR             598..778
                     /label=5' LTR (truncated)
                     /note="truncated 5' long terminal repeat (LTR) from HIV-1"
     misc_feature    825..950
                     /label=HIV-1 Psi
                     /note="packaging signal of human immunodeficiency virus 
                     type 1"
     misc_feature    1443..1676
                     /label=RRE
                     /note="The Rev response element (RRE) of HIV-1 allows for 
                     Rev-dependent mRNA export from the nucleus to the 
                     cytoplasm."
     CDS             1861..1905
                     /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    2203..2320
                     /label=cPPT/CTS
                     /note="central polypurine tract and central termination 
                     sequence of HIV-1"
     protein_bind    2364..2397
                     /label=loxP
                     /bound_moiety="Cre recombinase"
                     /note="Cre-mediated recombination occurs in the 8-bp core 
                     sequence (GCATACAT)."
     misc_RNA        complement(2408..2483)
                     /label=gRNA scaffold
                     /note="guide RNA scaffold for the Streptococcus pyogenes 
                     CRISPR/Cas9 system"
     promoter        complement(2508..2748)
                     /label=U6 promoter
                     /note="RNA polymerase III promoter for human U6 snRNA"
     primer_bind     complement(2558..2577)
                     /label=LKO.1 5'
                     /note="Human U6 promoter, forward primer"
     primer_bind     complement(2728..2748)
                     /label=hU6-F
                     /note="Human U6 promoter, forward primer"
     promoter        2780..3991
                     /label=UbC promoter
                     /note="human ubiquitin C promoter"
     primer_bind     3828..3847
                     /label=hUBCpro-F
                     /note="Human Ubiquitin C (UbC) promoter, forward primer"
     regulatory      4020..4029
                     /regulatory_class="other"
                     /note="vertebrate consensus sequence for strong initiation 
                     of translation (Kozak, 1987)"
     CDS             4029..4094
                     /codon_start=1
                     /product="three tandem FLAG(R) epitope tags, followed by an
                     enterokinase cleavage site"
                     /label=3xFLAG
                     /translation="DYKDHDGDYKDHDIDYKDDDDK"
     CDS             4110..4256
                     /codon_start=1
                     /product="tetrameric repeat of the minimal activation 
                     domain of herpes simplex virus VP16 (Beerli et al., 1998)"
                     /label=VP64
                     /translation="DALDDFDLDMLGSDALDDFDLDMLGSDALDDFDLDMLGSDALDDF
                     DLDM"
     CDS             4263..4283
                     /codon_start=1
                     /product="nuclear localization signal of SV40 (simian virus
                     40) large T antigen"
                     /label=SV40 NLS
                     /translation="PKKKRKV"
     CDS             4293..8396
                     /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             8409..8429
                     /codon_start=1
                     /product="nuclear localization signal of SV40 (simian virus
                     40) large T antigen"
                     /label=SV40 NLS
                     /translation="PKKKRKV"
     CDS             8442..8591
                     /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             8601..8654
                     /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             8661..9374
                     /codon_start=1
                     /product="enhanced GFP"
                     /label=EGFP
                     /translation="VSKGEELFTGVVPILVELDGDVNGHKFSVSGEGEGDATYGKLTLK
                     FICTTGKLPVPWPTLVTTLTYGVQCFSRYPDHMKQHDFFKSAMPEGYVQERTIFFKDDG
                     NYKTRAEVKFEGDTLVNRIELKGIDFKEDGNILGHKLEYNYNSHNVYIMADKQKNGIKV
                     NFKIRHNIEDGSVQLADHYQQNTPIGDGPVLLPDNHYLSTQSALSKDPNEKRDHMVLLE
                     FVTAAGITLGMDELYK"
     CDS             8661..9374
                     /codon_start=1
                     /product="the original enhanced GFP (Yang et al., 1996)"
                     /label=EGFP
                     /note="mammalian codon-optimized"
                     /translation="VSKGEELFTGVVPILVELDGDVNGHKFSVSGEGEGDATYGKLTLK
                     FICTTGKLPVPWPTLVTTLTYGVQCFSRYPDHMKQHDFFKSAMPEGYVQERTIFFKDDG
                     NYKTRAEVKFEGDTLVNRIELKGIDFKEDGNILGHKLEYNYNSHNVYIMADKQKNGIKV
                     NFKIRHNIEDGSVQLADHYQQNTPIGDGPVLLPDNHYLSTQSALSKDPNEKRDHMVLLE
                     FVTAAGITLGMDELYK"
     primer_bind     complement(8703..8724)
                     /label=EGFP-N
                     /note="EGFP, reverse primer"
     primer_bind     complement(8964..8983)
                     /label=EXFP-R
                     /note="For distinguishing EGFP variants, reverse primer"
     primer_bind     9311..9332
                     /label=EGFP-C
                     /note="EGFP, forward primer"
     protein_bind    9390..9423
                     /label=loxP
                     /bound_moiety="Cre recombinase"
                     /note="Cre-mediated recombination occurs in the 8-bp core 
                     sequence (GCATACAT)."
     misc_feature    9448..10036
                     /label=WPRE
                     /note="woodchuck hepatitis virus posttranscriptional 
                     regulatory element"
     primer_bind     complement(9501..9521)
                     /label=WPRE-R
                     /note="WPRE, reverse primer"
     CDS             complement(9919..9930)
                     /codon_start=1
                     /product="Factor Xa recognition and cleavage site"
                     /label=Factor Xa site
                     /translation="IEGR"
     primer_bind     complement(10039..10055)
                     /label=KS primer
                     /note="common sequencing primer, one of multiple similar 
                     variants"
     primer_bind     complement(10040..10056)
                     /label=pBluescriptKS
                     /note="For pBluescript vector"
     LTR             10561..10741
                     /label=5' LTR (truncated)
                     /note="truncated 5' long terminal repeat (LTR) from HIV-1"
     primer_bind     complement(10767..10784)
                     /label=BGH-rev
                     /note="Bovine growth hormone terminator, reverse primer. 
                     Also called BGH reverse"
     polyA_signal    10773..10997
                     /label=bGH poly(A) signal
                     /note="bovine growth hormone polyadenylation signal"
     rep_origin      11043..11471
                     /direction=RIGHT
                     /label=f1 ori
                     /note="f1 bacteriophage origin of replication; arrow 
                     indicates direction of (+) strand synthesis"
     primer_bind     complement(11130..11149)
                     /label=F1ori-R
                     /note="F1 origin, reverse primer"
     primer_bind     11340..11361
                     /label=F1ori-F
                     /note="F1 origin, forward primer"
     primer_bind     complement(11480..11500)
                     /label=pBABE 3'
                     /note="SV40 enhancer, reverse primer for pBABE vectors"
     promoter        11485..11814
                     /label=SV40 promoter
                     /note="SV40 enhancer and early promoter"
     rep_origin      11665..11800
                     /label=SV40 ori
                     /note="SV40 origin of replication"
     primer_bind     11727..11746
                     /label=SV40pro-F
                     /note="SV40 promoter/origin, forward primer"
     promoter        11862..11909
                     /label=EM7 promoter
                     /note="synthetic bacterial promoter "
     CDS             11928..12302
                     /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    12432..12553
                     /label=SV40 poly(A) signal
                     /note="SV40 polyadenylation signal"
     primer_bind     complement(12469..12488)
                     /label=SV40pA-R
                     /note="SV40 polyA, reverse primer"
     primer_bind     12523..12542
                     /label=EBV-rev
                     /note="SV40 polyA terminator, reverse primer"
     primer_bind     complement(12602..12618)
                     /label=M13 rev
                     /note="common sequencing primer, one of multiple similar 
                     variants"
     primer_bind     complement(12602..12618)
                     /label=M13 Reverse
                     /note="In lacZ gene. Also called M13-rev"
     primer_bind     complement(12615..12637)
                     /label=M13/pUC Reverse
                     /note="In lacZ gene"
     protein_bind    12626..12642
                     /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(12650..12680)
                     /label=lac promoter
                     /note="promoter for the E. coli lac operon"
     protein_bind    12695..12716
                     /label=CAP binding site
                     /bound_moiety="E. coli catabolite activator protein"
                     /note="CAP binding activates transcription in the presence 
                     of cAMP."
     primer_bind     complement(12833..12850)
                     /label=L4440
                     /note="L4440 vector, forward primer"
     rep_origin      complement(13004..13592)
                     /direction=LEFT
                     /label=ori
                     /note="high-copy-number ColE1/pMB1/pBR322/pUC origin of 
                     replication"
     primer_bind     complement(13084..13103)
                     /label=pBR322ori-F
                     /note="pBR322 origin, forward primer"
     CDS             complement(13763..14623)
                     /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     14386..14405
                     /label=Amp-R
                     /note="Ampicillin resistance gene, reverse primer"
     promoter        complement(14624..14728)
                     /gene="bla"
                     /label=AmpR promoter
     primer_bind     complement(14803..14822)
                     /label=pRS-marker
                     /note="pRS vectors, use to sequence yeast selectable 
                     marker"
ORIGIN
        1 gacattgatt attgactagt tattaatagt aatcaattac ggggtcatta gttcatagcc
       61 catatatgga gttccgcgtt acataactta cggtaaatgg cccgcctggc tgaccgccca
      121 acgacccccg cccattgacg tcaataatga cgtatgttcc catagtaacg ccaataggga
      181 ctttccattg acgtcaatgg gtggagtatt tacggtaaac tgcccacttg gcagtacatc
      241 aagtgtatca tatgccaagt acgcccccta ttgacgtcaa tgacggtaaa tggcccgcct
      301 ggcattatgc ccagtacatg accttatggg actttcctac ttggcagtac atctacgtat
      361 tagtcatcgc tattaccatg gtgatgcggt tttggcagta catcaatggg cgtggatagc
      421 ggtttgactc acggggattt ccaagtctcc accccattga cgtcaatggg agtttgtttt
      481 ggcaccaaaa tcaacgggac tttccaaaat gtcgtaacaa ctccgcccca ttgacgcaaa
      541 tgggcggtag gcgtgtacgg tgggaggtct atataagcag cgcgttttgc ctgtactggg
      601 tctctctggt tagaccagat ctgagcctgg gagctctctg gctaactagg gaacccactg
      661 cttaagcctc aataaagctt gccttgagtg cttcaagtag tgtgtgcccg tctgttgtgt
      721 gactctggta actagagatc cctcagaccc ttttagtcag tgtggaaaat ctctagcagt
      781 ggcgcccgaa cagggacttg aaagcgaaag ggaaaccaga ggagctctct cgacgcagga
      841 ctcggcttgc tgaagcgcgc acggcaagag gcgaggggcg gcgactggtg agtacgccaa
      901 aaattttgac tagcggaggc tagaaggaga gagatgggtg cgagagcgtc agtattaagc
      961 gggggagaat tagatcgcga tgggaaaaaa ttcggttaag gccaggggga aagaaaaaat
     1021 ataaattaaa acatatagta tgggcaagca gggagctaga acgattcgca gttaatcctg
     1081 gcctgttaga aacatcagaa ggctgtagac aaatactggg acagctacaa ccatcccttc
     1141 agacaggatc agaagaactt agatcattat ataatacagt agcaaccctc tattgtgtgc
     1201 atcaaaggat agagataaaa gacaccaagg aagctttaga caagatagag gaagagcaaa
     1261 acaaaagtaa gaccaccgca cagcaagcgg ccgctgatct tcagacctgg aggaggagat
     1321 atgagggaca attggagaag tgaattatat aaatataaag tagtaaaaat tgaaccatta
     1381 ggagtagcac ccaccaaggc aaagagaaga gtggtgcaga gagaaaaaag agcagtggga
     1441 ataggagctt tgttccttgg gttcttggga gcagcaggaa gcactatggg cgcagcgtca
     1501 atgacgctga cggtacaggc cagacaatta ttgtctggta tagtgcagca gcagaacaat
     1561 ttgctgaggg ctattgaggc gcaacagcat ctgttgcaac tcacagtctg gggcatcaag
     1621 cagctccagg caagaatcct ggctgtggaa agatacctaa aggatcaaca gctcctgggg
     1681 atttggggtt gctctggaaa actcatttgc accactgctg tgccttggaa tgctagttgg
     1741 agtaataaat ctctggaaca gatttggaat cacacgacct ggatggagtg ggacagagaa
     1801 attaacaatt acacaagctt aatacactcc ttaattgaag aatcgcaaaa ccagcaagaa
     1861 aagaatgaac aagaattatt ggaattagat aaatgggcaa gtttgtggaa ttggtttaac
     1921 ataacaaatt ggctgtggta tataaaatta ttcataatga tagtaggagg cttggtaggt
     1981 ttaagaatag tttttgctgt actttctata gtgaatagag ttaggcaggg atattcacca
     2041 ttatcgtttc agacccacct cccaaccccg aggggacccg acaggcccga aggaatagaa
     2101 gaagaaggtg gagagagaga cagagacaga tccattcgat tagtgaacgg atcggcactg
     2161 cgtgcgccaa ttctgcagac aaatggcagt attcatccac aattttaaaa gaaaaggggg
     2221 gattgggggg tacagtgcag gggaaagaat agtagacata atagcaacag acatacaaac
     2281 taaagaatta caaaaacaaa ttacaaaaat tcaaaatttt cgggtttatt acagggacag
     2341 cagagatcca gtttggttaa ttaataactt cgtatagcat acattatacg aagttattaa
     2401 aaaaaaagca ccgactcggt gccacttttt caagttgata acggactagc cttattttaa
     2461 cttgctattt ctagctctaa aactgagacg tccgtctccg gtgtttcgtc ctttccacaa
     2521 gatatataaa gccaagaaat cgaaatactt tcaagttacg gtaagcatat gatagtccat
     2581 tttaaaacat aattttaaaa ctgcaaacta cccaagaaat tattactttc tacgtcacgt
     2641 attttgtact aatatctttg tgtttacagt caaattaatt ccaattatct ctctaacagc
     2701 cttgtatcgt atatgcaaat atgaaggaat catgggaaat aggccctctt attaattaac
     2761 ccgtgtcggc tccagatctg gcctccgcgc cgggttttgg cgcctcccgc gggcgccccc
     2821 ctcctcacgg cgagcgctgc cacgtcagac gaagggcgca gcgagcgtcc tgatccttcc
     2881 gcccggacgc tcaggacagc ggcccgctgc tcataagact cggccttaga accccagtat
     2941 cagcagaagg acattttagg acgggacttg ggtgactcta gggcactggt tttctttcca
     3001 gagagcggaa caggcgagga aaagtagtcc cttctcggcg attctgcgga gggatctccg
     3061 tggggcggtg aacgccgatg attatataag gacgcgccgg gtgtggcaca gctagttccg
     3121 tcgcagccgg gatttgggtc gcggttcttg tttgtggatc gctgtgatcg tcacttggtg
     3181 agtagcgggc tgctgggctg gccggggctt tcgtggccgc cgggccgctc ggtgggacgg
     3241 aagcgtgtgg agagaccgcc aagggctgta gtctgggtcc gcgagcaagg ttgccctgaa
     3301 ctgggggttg gggggagcgc agcaaaatgg cggctgttcc cgagtcttga atggaagacg
     3361 cttgtgaggc gggctgtgag gtcgttgaaa caaggtgggg ggcatggtgg gcggcaagaa
     3421 cccaaggtct tgaggccttc gctaatgcgg gaaagctctt attcgggtga gatgggctgg
     3481 ggcaccatct ggggaccctg acgtgaagtt tgtcactgac tggagaactc ggtttgtcgt
     3541 ctgttgcggg ggcggcagtt atggcggtgc cgttgggcag tgcacccgta cctttgggag
     3601 cgcgcgccct cgtcgtgtcg tgacgtcacc cgttctgttg gcttataatg cagggtgggg
     3661 ccacctgccg gtaggtgtgc ggtaggcttt tctccgtcgc aggacgcagg gttcgggcct
     3721 agggtaggct ctcctgaatc gacaggcgcc ggacctctgg tgaggggagg gataagtgag
     3781 gcgtcagttt ctttggtcgg ttttatgtac ctatcttctt aagtagctga agctccggtt
     3841 ttgaactatg cgctcggggt tggcgagtgt gttttgtgaa gttttttagg caccttttga
     3901 aatgtaatca tttgggtcaa tatgtaattt tcagtgttag actagtaaat tgtccgctaa
     3961 attctggccg tttttggctt ttttgttaga cgaagcttgg gctgcaggtc gactctagag
     4021 ccaccatgga ctacaaagac catgacggtg attataaaga tcatgacatc gattacaagg
     4081 atgacgatga caagcacggt ccgcgggctg acgcattgga cgattttgat ctggatatgc
     4141 tgggaagtga cgccctcgat gattttgacc ttgacatgct tggttcggat gcccttgatg
     4201 actttgacct cgacatgctc ggcagtgacg cccttgatga tttcgacctg gacatggtta
     4261 accccaagaa gaagaggaag gtgggccgcg gaatggacaa gaagtactcc attgggctcg
     4321 ccatcggcac aaacagcgtc ggctgggccg tcattacgga cgagtacaag gtgccgagca
     4381 aaaaattcaa agttctgggc aataccgatc gccacagcat aaagaagaac ctcattggcg
     4441 ccctcctgtt cgactccggg gaaaccgccg aagccacgcg gctcaaaaga acagcacggc
     4501 gcagatatac ccgcagaaag aatcggatct gctacctgca ggagatcttt agtaatgaga
     4561 tggctaaggt ggatgactct ttcttccata ggctggagga gtcctttttg gtggaggagg
     4621 ataaaaagca cgagcgccac ccaatctttg gcaatatcgt ggacgaggtg gcgtaccatg
     4681 aaaagtaccc aaccatatat catctgagga agaagcttgt agacagtact gataaggctg
     4741 acttgcggtt gatctatctc gcgctggcgc atatgatcaa atttcgggga cacttcctca
     4801 tcgaggggga cctgaaccca gacaacagcg atgtcgacaa actctttatc caactggttc
     4861 agacttacaa tcagcttttc gaagagaacc cgatcaacgc atccggagtt gacgccaaag
     4921 caatcctgag cgctaggctg tccaaatccc ggcggctcga aaacctcatc gcacagctcc
     4981 ctggggagaa gaagaacggc ctgtttggta atcttatcgc cctgtcactc gggctgaccc
     5041 ccaactttaa atctaacttc gacctggccg aagatgccaa gcttcaactg agcaaagaca
     5101 cctacgatga tgatctcgac aatctgctgg cccagatcgg cgaccagtac gcagaccttt
     5161 ttttggcggc aaagaacctg tcagacgcca ttctgctgag tgatattctg cgagtgaaca
     5221 cggagatcac caaagctccg ctgagcgcta gtatgatcaa gcgctatgat gagcaccacc
     5281 aagacttgac tttgctgaag gcccttgtca gacagcaact gcctgagaag tacaaggaaa
     5341 ttttcttcga tcagtctaaa aatggctacg ccggatacat tgacggcgga gcaagccagg
     5401 aggaatttta caaatttatt aagcccatct tggaaaaaat ggacggcacc gaggagctgc
     5461 tggtaaagct taacagagaa gatctgttgc gcaaacagcg cactttcgac aatggaagca
     5521 tcccccacca gattcacctg ggcgaactgc acgctatcct caggcggcaa gaggatttct
     5581 accccttttt gaaagataac agggaaaaga ttgagaaaat cctcacattt cggataccct
     5641 actatgtagg ccccctcgcc cggggaaatt ccagattcgc gtggatgact cgcaaatcag
     5701 aagagaccat cactccctgg aacttcgagg aagtcgtgga taagggggcc tctgcccagt
     5761 ccttcatcga aaggatgact aactttgata aaaatctgcc taacgaaaag gtgcttccta
     5821 aacactctct gctgtacgag tacttcacag tttataacga gctcaccaag gtcaaatacg
     5881 tcacagaagg gatgagaaag ccagcattcc tgtctggaga gcagaagaaa gctatcgtgg
     5941 acctcctctt caagacgaac cggaaagtta ccgtgaaaca gctcaaagaa gactatttca
     6001 aaaagattga atgtttcgac tctgttgaaa tcagcggagt ggaggatcgc ttcaacgcat
     6061 ccctgggaac gtatcacgat ctcctgaaaa tcattaaaga caaggacttc ctggacaatg
     6121 aggagaacga ggacattctt gaggacattg tcctcaccct tacgttgttt gaagataggg
     6181 agatgattga agaacgcttg aaaacttacg ctcatctctt cgacgacaaa gtcatgaaac
     6241 agctcaagag gcgccgatat acaggatggg ggcggctgtc aagaaaactg atcaatggga
     6301 tccgagacaa gcagagtgga aagacaatcc tggattttct taagtccgat ggatttgcca
     6361 accggaactt catgcagttg atccatgatg actctctcac ctttaaggag gacatccaga
     6421 aagcacaagt ttctggccag ggggacagtc ttcacgagca catcgctaat cttgcaggta
     6481 gcccagctat caaaaaggga atactgcaga ccgttaaggt cgtggatgaa ctcgtcaaag
     6541 taatgggaag gcataagccc gagaatatcg ttatcgagat ggcccgagag aaccaaacta
     6601 cccagaaggg acagaagaac agtagggaaa ggatgaagag gattgaagag ggtataaaag
     6661 aactggggtc ccaaatcctt aaggaacacc cagttgaaaa cacccagctt cagaatgaga
     6721 agctctacct gtactacctg cagaacggca gggacatgta cgtggatcag gaactggaca
     6781 tcaatcggct ctccgactac gacgtggatg ccatcgtgcc ccagtctttt ctcaaagatg
     6841 attctattga taataaagtg ttgacaagat ccgataaaaa tagagggaag agtgataacg
     6901 tcccctcaga agaagttgtc aagaaaatga aaaattattg gcggcagctg ctgaacgcca
     6961 aactgatcac acaacggaag ttcgataatc tgactaaggc tgaacgaggt ggcctgtctg
     7021 agttggataa agccggcttc atcaaaaggc agcttgttga gacacgccag atcaccaagc
     7081 acgtggccca aattctcgat tcacgcatga acaccaagta cgatgaaaat gacaaactga
     7141 ttcgagaggt gaaagttatt actctgaagt ctaagctggt ctcagatttc agaaaggact
     7201 ttcagtttta taaggtgaga gagatcaaca attaccacca tgcgcatgat gcctacctga
     7261 atgcagtggt aggcactgca cttatcaaaa aatatcccaa gcttgaatct gaatttgttt
     7321 acggagacta taaagtgtac gatgttagga aaatgatcgc aaagtctgag caggaaatag
     7381 gcaaggccac cgctaagtac ttcttttaca gcaatattat gaattttttc aagaccgaga
     7441 ttacactggc caatggagag attcggaagc gaccacttat cgaaacaaac ggagaaacag
     7501 gagaaatcgt gtgggacaag ggtagggatt tcgcgacagt ccggaaggtc ctgtccatgc
     7561 cgcaggtgaa catcgttaaa aagaccgaag tacagaccgg aggcttctcc aaggaaagta
     7621 tcctcccgaa aaggaacagc gacaagctga tcgcacgcaa aaaagattgg gaccccaaga
     7681 aatacggcgg attcgattct cctacagtcg cttacagtgt actggttgtg gccaaagtgg
     7741 agaaagggaa gtctaaaaaa ctcaaaagcg tcaaggaact gctgggcatc acaatcatgg
     7801 agcgatcaag cttcgaaaaa aaccccatcg actttctcga ggcgaaagga tataaagagg
     7861 tcaaaaaaga cctcatcatt aagcttccca agtactctct ctttgagctt gaaaacggcc
     7921 ggaaacgaat gctcgctagt gcgggcgagc tgcagaaagg taacgagctg gcactgccct
     7981 ctaaatacgt taatttcttg tatctggcca gccactatga aaagctcaaa gggtctcccg
     8041 aagataatga gcagaagcag ctgttcgtgg aacaacacaa acactacctt gatgagatca
     8101 tcgagcaaat aagcgaattc tccaaaagag tgatcctcgc cgacgctaac ctcgataagg
     8161 tgctttctgc ttacaataag cacagggata agcccatcag ggagcaggca gaaaacatta
     8221 tccacttgtt tactctgacc aacttgggcg cgcctgcagc cttcaagtac ttcgacacca
     8281 ccatagacag aaagcggtac acctctacaa aggaggtcct ggacgccaca ctgattcatc
     8341 agtcaattac ggggctctat gaaacaagaa tcgacctctc tcagctcggt ggagacagca
     8401 gggctgaccc caagaagaag aggaaggtgg ctagccgcgc cgacgcgctg gacgatttcg
     8461 atctcgacat gctgggttct gatgccctcg atgactttga cctggatatg ttgggaagcg
     8521 acgcattgga tgactttgat ctggacatgc tcggctccga tgctctggac gatttcgatc
     8581 tcgatatgtt aatcgctagc gagggcagag gaagtcttct aacatgcggt gacgtggagg
     8641 agaatcccgg ccctggtacc gtgagcaagg gcgaggagct gttcaccggg gtggtgccca
     8701 tcctggtcga gctggacggc gacgtaaacg gccacaagtt cagcgtgtcc ggcgagggcg
     8761 agggcgatgc cacctacggc aagctgaccc tgaagttcat ctgcaccacc ggcaagctgc
     8821 ccgtgccctg gcccaccctc gtgaccaccc tgacctacgg cgtgcagtgc ttcagccgct
     8881 accccgacca catgaagcag cacgacttct tcaagtccgc catgcccgaa ggctacgtcc
     8941 aggagcgcac catcttcttc aaggacgacg gcaactacaa gacccgcgcc gaggtgaagt
     9001 tcgagggcga caccctggtg aaccgcatcg agctgaaggg catcgacttc aaggaggacg
     9061 gcaacatcct ggggcacaag ctggagtaca actacaacag ccacaacgtc tatatcatgg
     9121 ccgacaagca gaagaacggc atcaaggtga acttcaagat ccgccacaac atcgaggacg
     9181 gcagcgtgca gctcgccgac cactaccagc agaacacccc catcggcgac ggccccgtgc
     9241 tgctgcccga caaccactac ctgagcaccc agtccgccct gagcaaagac cccaacgaga
     9301 agcgcgatca catggtcctg ctggagttcg tgaccgccgc cgggatcact ctcggcatgg
     9361 acgagctgta caagaccggt tgataataga taacttcgta tagcatacat tatacgaagt
     9421 tatgaattcg atatcaagct tatcgataat caacctctgg attacaaaat ttgtgaaaga
     9481 ttgactggta ttcttaacta tgttgctcct tttacgctat gtggatacgc tgctttaatg
     9541 cctttgtatc atgctattgc ttcccgtatg gctttcattt tctcctcctt gtataaatcc
     9601 tggttgctgt ctctttatga ggagttgtgg cccgttgtca ggcaacgtgg cgtggtgtgc
     9661 actgtgtttg ctgacgcaac ccccactggt tggggcattg ccaccacctg tcagctcctt
     9721 tccgggactt tcgctttccc cctccctatt gccacggcgg aactcatcgc cgcctgcctt
     9781 gcccgctgct ggacaggggc tcggctgttg ggcactgaca attccgtggt gttgtcgggg
     9841 aaatcatcgt cctttccttg gctgctcgcc tgtgttgcca cctggattct gcgcgggacg
     9901 tccttctgct acgtcccttc ggccctcaat ccagcggacc ttccttcccg cggcctgctg
     9961 ccggctctgc ggcctcttcc gcgtcttcgc cttcgccctc agacgagtcg gatctccctt
    10021 tgggccgcct ccccgcatcg ataccgtcga cctcgagacc tagaaaaaca tggagcaatc
    10081 acaagtagca atacagcagc taccaatgct gattgtgcct ggctagaagc acaagaggag
    10141 gaggaggtgg gttttccagt cacacctcag gtacctttaa gaccaatgac ttacaaggca
    10201 gctgtagatc ttagccactt tttaaaagaa aaggggggac tggaagggct aattcactcc
    10261 caacgaagac aagatatcct tgatctgtgg atctaccaca cacaaggcta cttccctgat
    10321 tggcagaact acacaccagg gccagggatc agatatccac tgacctttgg atggtgctac
    10381 aagctagtac cagttgagca agagaaggta gaagaagcca atgaaggaga gaacacccgc
    10441 ttgttacacc ctgtgagcct gcatgggatg gatgacccgg agagagaagt attagagtgg
    10501 aggtttgaca gccgcctagc atttcatcac atggcccgag agctgcatcc ggactgtact
    10561 gggtctctct ggttagacca gatctgagcc tgggagctct ctggctaact agggaaccca
    10621 ctgcttaagc ctcaataaag cttgccttga gtgcttcaag tagtgtgtgc ccgtctgttg
    10681 tgtgactctg gtaactagag atccctcaga cccttttagt cagtgtggaa aatctctagc
    10741 agggcccgtt taaacccgct gatcagcctc gactgtgcct tctagttgcc agccatctgt
    10801 tgtttgcccc tcccccgtgc cttccttgac cctggaaggt gccactccca ctgtcctttc
    10861 ctaataaaat gaggaaattg catcgcattg tctgagtagg tgtcattcta ttctgggggg
    10921 tggggtgggg caggacagca agggggagga ttgggaagac aatagcaggc atgctgggga
    10981 tgcggtgggc tctatggctt ctgaggcgga aagaaccagc tggggctcta gggggtatcc
    11041 ccacgcgccc tgtagcggcg cattaagcgc ggcgggtgtg gtggttacgc gcagcgtgac
    11101 cgctacactt gccagcgccc tagcgcccgc tcctttcgct ttcttccctt cctttctcgc
    11161 cacgttcgcc ggctttcccc gtcaagctct aaatcggggg ctccctttag ggttccgatt
    11221 tagtgcttta cggcacctcg accccaaaaa acttgattag ggtgatggtt cacgtagtgg
    11281 gccatcgccc tgatagacgg tttttcgccc tttgacgttg gagtccacgt tctttaatag
    11341 tggactcttg ttccaaactg gaacaacact caaccctatc tcggtctatt cttttgattt
    11401 ataagggatt ttgccgattt cggcctattg gttaaaaaat gagctgattt aacaaaaatt
    11461 taacgcgaat taattctgtg gaatgtgtgt cagttagggt gtggaaagtc cccaggctcc
    11521 ccagcaggca gaagtatgca aagcatgcat ctcaattagt cagcaaccag gtgtggaaag
    11581 tccccaggct ccccagcagg cagaagtatg caaagcatgc atctcaatta gtcagcaacc
    11641 atagtcccgc ccctaactcc gcccatcccg cccctaactc cgcccagttc cgcccattct
    11701 ccgccccatg gctgactaat tttttttatt tatgcagagg ccgaggccgc ctctgcctct
    11761 gagctattcc agaagtagtg aggaggcttt tttggaggcc taggcttttg caaaaagctc
    11821 ccgggagctt gtatatccat tttcggatct gatcagcacg tgttgacaat taatcatcgg
    11881 catagtatat cggcatagta taatacgaca aggtgaggaa ctaaaccatg gccaagttga
    11941 ccagtgccgt tccggtgctc accgcgcgcg acgtcgccgg agcggtcgag ttctggaccg
    12001 accggctcgg gttctcccgg gacttcgtgg aggacgactt cgccggtgtg gtccgggacg
    12061 acgtgaccct gttcatcagc gcggtccagg accaggtggt gccggacaac accctggcct
    12121 gggtgtgggt gcgcggcctg gacgagctgt acgccgagtg gtcggaggtc gtgtccacga
    12181 acttccggga cgcctccggg ccggccatga ccgagatcgg cgagcagccg tgggggcggg
    12241 agttcgccct gcgcgacccg gccggcaact gcgtgcactt cgtggccgag gagcaggact
    12301 gacacgtgct acgagatttc gattccaccg ccgccttcta tgaaaggttg ggcttcggaa
    12361 tcgttttccg ggacgccggc tggatgatcc tccagcgcgg ggatctcatg ctggagttct
    12421 tcgcccaccc caacttgttt attgcagctt ataatggtta caaataaagc aatagcatca
    12481 caaatttcac aaataaagca tttttttcac tgcattctag ttgtggtttg tccaaactca
    12541 tcaatgtatc ttatcatgtc tgtataccgt cgacctctag ctagagcttg gcgtaatcat
    12601 ggtcatagct gtttcctgtg tgaaattgtt atccgctcac aattccacac aacatacgag
    12661 ccggaagcat aaagtgtaaa gcctggggtg cctaatgagt gagctaactc acattaattg
    12721 cgttgcgctc actgcccgct ttccagtcgg gaaacctgtc gtgccagctg cattaatgaa
    12781 tcggccaacg cgcggggaga ggcggtttgc gtattgggcg ctcttccgct tcctcgctca
    12841 ctgactcgct gcgctcggtc gttcggctgc ggcgagcggt atcagctcac tcaaaggcgg
    12901 taatacggtt atccacagaa tcaggggata acgcaggaaa gaacatgtga gcaaaaggcc
    12961 agcaaaaggc caggaaccgt aaaaaggccg cgttgctggc gtttttccat aggctccgcc
    13021 cccctgacga gcatcacaaa aatcgacgct caagtcagag gtggcgaaac ccgacaggac
    13081 tataaagata ccaggcgttt ccccctggaa gctccctcgt gcgctctcct gttccgaccc
    13141 tgccgcttac cggatacctg tccgcctttc tcccttcggg aagcgtggcg ctttctcata
    13201 gctcacgctg taggtatctc agttcggtgt aggtcgttcg ctccaagctg ggctgtgtgc
    13261 acgaaccccc cgttcagccc gaccgctgcg ccttatccgg taactatcgt cttgagtcca
    13321 acccggtaag acacgactta tcgccactgg cagcagccac tggtaacagg attagcagag
    13381 cgaggtatgt aggcggtgct acagagttct tgaagtggtg gcctaactac ggctacacta
    13441 gaagaacagt atttggtatc tgcgctctgc tgaagccagt taccttcgga aaaagagttg
    13501 gtagctcttg atccggcaaa caaaccaccg ctggtagcgg tggttttttt gtttgcaagc
    13561 agcagattac gcgcagaaaa aaaggatctc aagaagatcc tttgatcttt tctacggggt
    13621 ctgacgctca gtggaacgaa aactcacgtt aagggatttt ggtcatgaga ttatcaaaaa
    13681 ggatcttcac ctagatcctt ttaaattaaa aatgaagttt taaatcaatc taaagtatat
    13741 atgagtaaac ttggtctgac agttaccaat gcttaatcag tgaggcacct atctcagcga
    13801 tctgtctatt tcgttcatcc atagttgcct gactccccgt cgtgtagata actacgatac
    13861 gggagggctt accatctggc cccagtgctg caatgatacc gcgagaccca cgctcaccgg
    13921 ctccagattt atcagcaata aaccagccag ccggaagggc cgagcgcaga agtggtcctg
    13981 caactttatc cgcctccatc cagtctatta attgttgccg ggaagctaga gtaagtagtt
    14041 cgccagttaa tagtttgcgc aacgttgttg ccattgctac aggcatcgtg gtgtcacgct
    14101 cgtcgtttgg tatggcttca ttcagctccg gttcccaacg atcaaggcga gttacatgat
    14161 cccccatgtt gtgcaaaaaa gcggttagct ccttcggtcc tccgatcgtt gtcagaagta
    14221 agttggccgc agtgttatca ctcatggtta tggcagcact gcataattct cttactgtca
    14281 tgccatccgt aagatgcttt tctgtgactg gtgagtactc aaccaagtca ttctgagaat
    14341 agtgtatgcg gcgaccgagt tgctcttgcc cggcgtcaat acgggataat accgcgccac
    14401 atagcagaac tttaaaagtg ctcatcattg gaaaacgttc ttcggggcga aaactctcaa
    14461 ggatcttacc gctgttgaga tccagttcga tgtaacccac tcgtgcaccc aactgatctt
    14521 cagcatcttt tactttcacc agcgtttctg ggtgagcaaa aacaggaagg caaaatgccg
    14581 caaaaaaggg aataagggcg acacggaaat gttgaatact catactcttc ctttttcaat
    14641 attattgaag catttatcag ggttattgtc tcatgagcgg atacatattt gaatgtattt
    14701 agaaaaataa acaaataggg gttccgcgca catttccccg aaaagtgcca cctgacgtcg
    14761 acggatcggg agatctcccg atcccctatg gtgcactctc agtacaatct gctctgatgc
    14821 cgcatagtta agccagtatc tgctccctgc ttgtgtgttg gaggtcgctg agtagtgcgc
    14881 gagcaaaatt taagctacaa caaggcaagg cttgaccgac aattgcatga agaatctgct
    14941 tagggttagg cgttttgcgc tgcttcgcga tgtacgggcc agatatacgc gtt
//