LOCUS       Exported               14937 bp ds-DNA     circular SYN 12-MAY-2021
DEFINITION  Co-expresses human optimized S. pyogenes dCas9-VP64 and GFP.
ACCESSION   .
VERSION     .
KEYWORDS    pLV hUbC-dCas9 VP64-T2A-GFP
SOURCE      synthetic DNA construct
  ORGANISM  synthetic DNA construct
REFERENCE   1  (bases 1 to 14937)
  AUTHORS   Kabadi AM, Ousterout DG, Hilton IB, Gersbach CA
  TITLE     Multiplex CRISPR/Cas9-based genome engineering from a single 
            lentiviral vector.
  JOURNAL   Nucleic Acids Res. 2014 Aug 13. pii: gku749.
  PUBMED    25122746
REFERENCE   2  (bases 1 to 14937)
  AUTHORS   .
  TITLE     Direct Submission
  JOURNAL   Exported May 12, 2021 from SnapGene Server 1.1.58
            http://www.snapgene.com
FEATURES             Location/Qualifiers
     source          1..14937
                     /organism="synthetic DNA construct"
                     /mol_type="other DNA"
     primer_bind     complement(47..66)
                     /label=pRS-marker
                     /note="pRS vectors, use to sequence yeast selectable 
                     marker"
     enhancer        238..617
                     /label=CMV enhancer
                     /note="human cytomegalovirus immediate early enhancer"
     promoter        619..817
                     /label=CMV promoter
                     /note="human cytomegalovirus (CMV) immediate early 
                     promoter"
     primer_bind     772..792
                     /label=CMV-F
                     /note="Human CMV immediate early promoter, forward primer"
     LTR             835..1015
                     /label=5' LTR (truncated)
                     /note="truncated 5' long terminal repeat (LTR) from HIV-1"
     misc_feature    1062..1187
                     /label=HIV-1 Psi
                     /note="packaging signal of human immunodeficiency virus 
                     type 1"
     misc_feature    1680..1913
                     /label=RRE
                     /note="The Rev response element (RRE) of HIV-1 allows for 
                     Rev-dependent mRNA export from the nucleus to the 
                     cytoplasm."
     CDS             2098..2142
                     /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    2440..2557
                     /label=cPPT/CTS
                     /note="central polypurine tract and central termination 
                     sequence of HIV-1"
     protein_bind    2602..2635
                     /label=loxP
                     /bound_moiety="Cre recombinase"
                     /note="Cre-mediated recombination occurs in the 8-bp core 
                     sequence (GCATACAT)."
     CDS             complement(2622..2948)
                     /codon_start=1
                     /gene="lacZ fragment"
                     /product="LacZ-alpha fragment of beta-galactosidase"
                     /label=lacZ-alpha
                     /translation="MTMITPSAQLTLTKGNKSWRTSRGGPVPNSPYSESYYARSLAVVL
                     QRRDWENPGVTQLNRLAAHPPFASWRNSEEARTDRPSQQLRSLNGEWDAPCSGATTVSY
                     HNFV"
     primer_bind     2798..2820
                     /label=M13/pUC Forward
                     /note="In lacZ gene"
     primer_bind     2812..2829
                     /label=M13 Forward
                     /note="In lacZ gene. Also called M13-F20 or M13 (-21) 
                     Forward"
     primer_bind     2813..2829
                     /label=M13 fwd
                     /note="common sequencing primer, one of multiple similar 
                     variants"
     primer_bind     2839..2858
                     /label=T7
                     /note="T7 promoter, forward primer"
     promoter        2839..2857
                     /label=T7 promoter
                     /note="promoter for bacteriophage T7 RNA polymerase"
     primer_bind     complement(2905..2925)
                     /label=T3
                     /note="T3 promoter, forward primer"
     promoter        complement(2905..2923)
                     /label=T3 promoter
                     /note="promoter for bacteriophage T3 RNA polymerase"
     primer_bind     complement(2944..2960)
                     /label=M13 rev
                     /note="common sequencing primer, one of multiple similar 
                     variants"
     protein_bind    2968..2984
                     /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(2992..3022)
                     /label=lac promoter
                     /note="promoter for the E. coli lac operon"
     protein_bind    3037..3058
                     /label=CAP binding site
                     /bound_moiety="E. coli catabolite activator protein"
                     /note="CAP binding activates transcription in the presence 
                     of cAMP."
     promoter        3124..4334
                     /label=UbC promoter
                     /note="human ubiquitin C promoter"
     primer_bind     4171..4190
                     /label=hUBCpro-F
                     /note="Human Ubiquitin C (UbC) promoter, forward primer"
     regulatory      4363..4372
                     /regulatory_class="other"
                     /note="vertebrate consensus sequence for strong initiation 
                     of translation (Kozak, 1987)"
     CDS             4372..4437
                     /codon_start=1
                     /product="three tandem FLAG(R) epitope tags, followed by an
                     enterokinase cleavage site"
                     /label=3xFLAG
                     /translation="DYKDHDGDYKDHDIDYKDDDDK"
     CDS             4444..4464
                     /codon_start=1
                     /product="nuclear localization signal of SV40 (simian virus
                     40) large T antigen"
                     /label=SV40 NLS
                     /translation="PKKKRKV"
     CDS             4474..8577
                     /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             8590..8610
                     /codon_start=1
                     /product="nuclear localization signal of SV40 (simian virus
                     40) large T antigen"
                     /label=SV40 NLS
                     /translation="PKKKRKV"
     CDS             8623..8772
                     /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             8782..8835
                     /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             8842..9555
                     /codon_start=1
                     /product="enhanced GFP"
                     /label=EGFP
                     /translation="VSKGEELFTGVVPILVELDGDVNGHKFSVSGEGEGDATYGKLTLK
                     FICTTGKLPVPWPTLVTTLTYGVQCFSRYPDHMKQHDFFKSAMPEGYVQERTIFFKDDG
                     NYKTRAEVKFEGDTLVNRIELKGIDFKEDGNILGHKLEYNYNSHNVYIMADKQKNGIKV
                     NFKIRHNIEDGSVQLADHYQQNTPIGDGPVLLPDNHYLSTQSALSKDPNEKRDHMVLLE
                     FVTAAGITLGMDELYK"
     CDS             8842..9555
                     /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(8884..8905)
                     /label=EGFP-N
                     /note="EGFP, reverse primer"
     primer_bind     complement(9145..9164)
                     /label=EXFP-R
                     /note="For distinguishing EGFP variants, reverse primer"
     primer_bind     9492..9513
                     /label=EGFP-C
                     /note="EGFP, forward primer"
     protein_bind    9571..9604
                     /label=loxP
                     /bound_moiety="Cre recombinase"
                     /note="Cre-mediated recombination occurs in the 8-bp core 
                     sequence (GCATACAT)."
     misc_feature    9629..10217
                     /label=WPRE
                     /note="woodchuck hepatitis virus posttranscriptional 
                     regulatory element"
     primer_bind     complement(9682..9702)
                     /label=WPRE-R
                     /note="WPRE, reverse primer"
     CDS             complement(10100..10111)
                     /codon_start=1
                     /product="Factor Xa recognition and cleavage site"
                     /label=Factor Xa site
                     /translation="IEGR"
     primer_bind     complement(10220..10236)
                     /label=KS primer
                     /note="common sequencing primer, one of multiple similar 
                     variants"
     primer_bind     complement(10221..10237)
                     /label=pBluescriptKS
                     /note="For pBluescript vector"
     LTR             10742..10922
                     /label=5' LTR (truncated)
                     /note="truncated 5' long terminal repeat (LTR) from HIV-1"
     primer_bind     complement(10948..10965)
                     /label=BGH-rev
                     /note="Bovine growth hormone terminator, reverse primer. 
                     Also called BGH reverse"
     polyA_signal    10954..11178
                     /label=bGH poly(A) signal
                     /note="bovine growth hormone polyadenylation signal"
     rep_origin      11224..11652
                     /direction=RIGHT
                     /label=f1 ori
                     /note="f1 bacteriophage origin of replication; arrow 
                     indicates direction of (+) strand synthesis"
     primer_bind     complement(11311..11330)
                     /label=F1ori-R
                     /note="F1 origin, reverse primer"
     primer_bind     11521..11542
                     /label=F1ori-F
                     /note="F1 origin, forward primer"
     primer_bind     complement(11661..11681)
                     /label=pBABE 3'
                     /note="SV40 enhancer, reverse primer for pBABE vectors"
     promoter        11666..11995
                     /label=SV40 promoter
                     /note="SV40 enhancer and early promoter"
     rep_origin      11846..11981
                     /label=SV40 ori
                     /note="SV40 origin of replication"
     primer_bind     11908..11927
                     /label=SV40pro-F
                     /note="SV40 promoter/origin, forward primer"
     promoter        12043..12090
                     /label=EM7 promoter
                     /note="synthetic bacterial promoter "
     CDS             12109..12483
                     /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    12613..12734
                     /label=SV40 poly(A) signal
                     /note="SV40 polyadenylation signal"
     primer_bind     complement(12650..12669)
                     /label=SV40pA-R
                     /note="SV40 polyA, reverse primer"
     primer_bind     12704..12723
                     /label=EBV-rev
                     /note="SV40 polyA terminator, reverse primer"
     primer_bind     complement(12783..12799)
                     /label=M13 rev
                     /note="common sequencing primer, one of multiple similar 
                     variants"
     protein_bind    12807..12823
                     /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(12831..12861)
                     /label=lac promoter
                     /note="promoter for the E. coli lac operon"
     protein_bind    12876..12897
                     /label=CAP binding site
                     /bound_moiety="E. coli catabolite activator protein"
                     /note="CAP binding activates transcription in the presence 
                     of cAMP."
     primer_bind     complement(13014..13031)
                     /label=L4440
                     /note="L4440 vector, forward primer"
     rep_origin      complement(13185..13773)
                     /direction=LEFT
                     /label=ori
                     /note="high-copy-number ColE1/pMB1/pBR322/pUC origin of 
                     replication"
     primer_bind     complement(13265..13284)
                     /label=pBR322ori-F
                     /note="pBR322 origin, forward primer"
     CDS             complement(13944..14804)
                     /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     14567..14586
                     /label=Amp-R
                     /note="Ampicillin resistance gene, reverse primer"
     promoter        complement(14805..14909)
                     /gene="bla"
                     /label=AmpR promoter
ORIGIN
        1 gtcgacggat cgggagatct cccgatcccc tatggtgcac tctcagtaca atctgctctg
       61 atgccgcata gttaagccag tatctgctcc ctgcttgtgt gttggaggtc gctgagtagt
      121 gcgcgagcaa aatttaagct acaacaaggc aaggcttgac cgacaattgc atgaagaatc
      181 tgcttagggt taggcgtttt gcgctgcttc gcgatgtacg ggccagatat acgcgttgac
      241 attgattatt gactagttat taatagtaat caattacggg gtcattagtt catagcccat
      301 atatggagtt ccgcgttaca taacttacgg taaatggccc gcctggctga ccgcccaacg
      361 acccccgccc attgacgtca ataatgacgt atgttcccat agtaacgcca atagggactt
      421 tccattgacg tcaatgggtg gagtatttac ggtaaactgc ccacttggca gtacatcaag
      481 tgtatcatat gccaagtacg ccccctattg acgtcaatga cggtaaatgg cccgcctggc
      541 attatgccca gtacatgacc ttatgggact ttcctacttg gcagtacatc tacgtattag
      601 tcatcgctat taccatggtg atgcggtttt ggcagtacat caatgggcgt ggatagcggt
      661 ttgactcacg gggatttcca agtctccacc ccattgacgt caatgggagt ttgttttggc
      721 accaaaatca acgggacttt ccaaaatgtc gtaacaactc cgccccattg acgcaaatgg
      781 gcggtaggcg tgtacggtgg gaggtctata taagcagcgc gttttgcctg tactgggtct
      841 ctctggttag accagatctg agcctgggag ctctctggct aactagggaa cccactgctt
      901 aagcctcaat aaagcttgcc ttgagtgctt caagtagtgt gtgcccgtct gttgtgtgac
      961 tctggtaact agagatccct cagacccttt tagtcagtgt ggaaaatctc tagcagtggc
     1021 gcccgaacag ggacttgaaa gcgaaaggga aaccagagga gctctctcga cgcaggactc
     1081 ggcttgctga agcgcgcacg gcaagaggcg aggggcggcg actggtgagt acgccaaaaa
     1141 ttttgactag cggaggctag aaggagagag atgggtgcga gagcgtcagt attaagcggg
     1201 ggagaattag atcgcgatgg gaaaaaattc ggttaaggcc agggggaaag aaaaaatata
     1261 aattaaaaca tatagtatgg gcaagcaggg agctagaacg attcgcagtt aatcctggcc
     1321 tgttagaaac atcagaaggc tgtagacaaa tactgggaca gctacaacca tcccttcaga
     1381 caggatcaga agaacttaga tcattatata atacagtagc aaccctctat tgtgtgcatc
     1441 aaaggataga gataaaagac accaaggaag ctttagacaa gatagaggaa gagcaaaaca
     1501 aaagtaagac caccgcacag caagcggccg ctgatcttca gacctggagg aggagatatg
     1561 agggacaatt ggagaagtga attatataaa tataaagtag taaaaattga accattagga
     1621 gtagcaccca ccaaggcaaa gagaagagtg gtgcagagag aaaaaagagc agtgggaata
     1681 ggagctttgt tccttgggtt cttgggagca gcaggaagca ctatgggcgc agcgtcaatg
     1741 acgctgacgg tacaggccag acaattattg tctggtatag tgcagcagca gaacaatttg
     1801 ctgagggcta ttgaggcgca acagcatctg ttgcaactca cagtctgggg catcaagcag
     1861 ctccaggcaa gaatcctggc tgtggaaaga tacctaaagg atcaacagct cctggggatt
     1921 tggggttgct ctggaaaact catttgcacc actgctgtgc cttggaatgc tagttggagt
     1981 aataaatctc tggaacagat ttggaatcac acgacctgga tggagtggga cagagaaatt
     2041 aacaattaca caagcttaat acactcctta attgaagaat cgcaaaacca gcaagaaaag
     2101 aatgaacaag aattattgga attagataaa tgggcaagtt tgtggaattg gtttaacata
     2161 acaaattggc tgtggtatat aaaattattc ataatgatag taggaggctt ggtaggttta
     2221 agaatagttt ttgctgtact ttctatagtg aatagagtta ggcagggata ttcaccatta
     2281 tcgtttcaga cccacctccc aaccccgagg ggacccgaca ggcccgaagg aatagaagaa
     2341 gaaggtggag agagagacag agacagatcc attcgattag tgaacggatc ggcactgcgt
     2401 gcgccaattc tgcagacaaa tggcagtatt catccacaat tttaaaagaa aaggggggat
     2461 tggggggtac agtgcagggg aaagaatagt agacataata gcaacagaca tacaaactaa
     2521 agaattacaa aaacaaatta caaaaattca aaattttcgg gtttattaca gggacagcag
     2581 agatccagtt tggttaatta aataacttcg tatagcatac attatacgaa gttatgataa
     2641 gagacggtgg tggcgccgct acagggcgcg tcccattcgc cattcaggct gcgcaactgt
     2701 tgggaagggc gatcggtgcg ggcctcttcg ctattacgcc agctggcgaa agggggatgt
     2761 gctgcaaggc gattaagttg ggtaacgcca gggttttccc agtcacgacg ttgtaaaacg
     2821 acggccagtg agcgcgcgta atacgactca ctatagggcg aattgggtac cgggcccccc
     2881 ctcgaggtcc tccagctttt gttcccttta gtgagggtta attgcgcgct tggcgtaatc
     2941 atggtcatag ctgtttcctg tgtgaaattg ttatccgctc acaattccac acaacatacg
     3001 agccggaagc ataaagtgta aagcctgggg tgcctaatga gtgagctaac tcacattaat
     3061 tgcgttgcgc tcactgcccg ctttccactg catgacgtct ccacaattaa ttaagggtgc
     3121 agcggcctcc gcgccgggtt ttggcgcctc ccgcgggcgc ccccctcctc acggcgagcg
     3181 ctgccacgtc agacgaaggg cgcaggagcg ttcctgatcc ttccgcccgg acgctcagga
     3241 cagcggcccg ctgctcataa gactcggcct tagaacccca gtatcagcag aaggacattt
     3301 taggacggga cttgggtgac tctagggcac tggttttctt tccagagagc ggaacaggcg
     3361 aggaaaagta gtcccttctc ggcgattctg cggagggatc tccgtggggc ggtgaacgcc
     3421 gatgattata taaggacgcg ccgggtgtgg cacagctagt tccgtcgcag ccgggatttg
     3481 ggtcgcggtt cttgtttgtg gatcgctgtg atcgtcactt ggtgagttgc gggctgctgg
     3541 gctggccggg gctttcgtgg ccgccgggcc gctcggtggg acggaagcgt gtggagagac
     3601 cgccaagggc tgtagtctgg gtccgcgagc aaggttgccc tgaactgggg gttgggggga
     3661 gcgcacaaaa tggcggctgt tcccgagtct tgaatggaag acgcttgtaa ggcgggctgt
     3721 gaggtcgttg aaacaaggtg gggggcatgg tgggcggcaa gaacccaagg tcttgaggcc
     3781 ttcgctaatg cgggaaagct cttattcggg tgagatgggc tggggcacca tctggggacc
     3841 ctgacgtgaa gtttgtcact gactggagaa ctcgggtttg tcgtctggtt gcgggggcgg
     3901 cagttatgcg gtgccgttgg gcagtgcacc cgtacctttg ggagcgcgcg cctcgtcgtg
     3961 tcgtgacgtc acccgttctg ttggcttata atgcagggtg gggccacctg ccggtaggtg
     4021 tgcggtaggc ttttctccgt cgcaggacgc agggttcggg cctagggtag gctctcctga
     4081 atcgacaggc gccggacctc tggtgagggg agggataagt gaggcgtcag tttctttggt
     4141 cggttttatg tacctatctt cttaagtagc tgaagctccg gttttgaact atgcgctcgg
     4201 ggttggcgag tgtgttttgt gaagtttttt aggcaccttt tgaaatgtaa tcatttgggt
     4261 caatatgtaa ttttcagtgt tagactagta aattgtccgc taaattctgg ccgtttttgg
     4321 cttttttgtt agacgaagct tgggctgcag gtcgactcta gagccaccat ggactacaaa
     4381 gaccatgacg gtgattataa agatcatgac atcgattaca aggatgacga tgacaagatg
     4441 gcccccaaga agaagaggaa ggtgggccgc ggaatggaca agaagtactc cattgggctc
     4501 gccatcggca caaacagcgt cggctgggcc gtcattacgg acgagtacaa ggtgccgagc
     4561 aaaaaattca aagttctggg caataccgat cgccacagca taaagaagaa cctcattggc
     4621 gccctcctgt tcgactccgg ggaaaccgcc gaagccacgc ggctcaaaag aacagcacgg
     4681 cgcagatata cccgcagaaa gaatcggatc tgctacctgc aggagatctt tagtaatgag
     4741 atggctaagg tggatgactc tttcttccat aggctggagg agtccttttt ggtggaggag
     4801 gataaaaagc acgagcgcca cccaatcttt ggcaatatcg tggacgaggt ggcgtaccat
     4861 gaaaagtacc caaccatata tcatctgagg aagaagcttg tagacagtac tgataaggct
     4921 gacttgcggt tgatctatct cgcgctggcg catatgatca aatttcgggg acacttcctc
     4981 atcgaggggg acctgaaccc agacaacagc gatgtcgaca aactctttat ccaactggtt
     5041 cagacttaca atcagctttt cgaagagaac ccgatcaacg catccggagt tgacgccaaa
     5101 gcaatcctga gcgctaggct gtccaaatcc cggcggctcg aaaacctcat cgcacagctc
     5161 cctggggaga agaagaacgg cctgtttggt aatcttatcg ccctgtcact cgggctgacc
     5221 cccaacttta aatctaactt cgacctggcc gaagatgcca agcttcaact gagcaaagac
     5281 acctacgatg atgatctcga caatctgctg gcccagatcg gcgaccagta cgcagacctt
     5341 tttttggcgg caaagaacct gtcagacgcc attctgctga gtgatattct gcgagtgaac
     5401 acggagatca ccaaagctcc gctgagcgct agtatgatca agcgctatga tgagcaccac
     5461 caagacttga ctttgctgaa ggcccttgtc agacagcaac tgcctgagaa gtacaaggaa
     5521 attttcttcg atcagtctaa aaatggctac gccggataca ttgacggcgg agcaagccag
     5581 gaggaatttt acaaatttat taagcccatc ttggaaaaaa tggacggcac cgaggagctg
     5641 ctggtaaagc ttaacagaga agatctgttg cgcaaacagc gcactttcga caatggaagc
     5701 atcccccacc agattcacct gggcgaactg cacgctatcc tcaggcggca agaggatttc
     5761 tacccctttt tgaaagataa cagggaaaag attgagaaaa tcctcacatt tcggataccc
     5821 tactatgtag gccccctcgc ccggggaaat tccagattcg cgtggatgac tcgcaaatca
     5881 gaagagacca tcactccctg gaacttcgag gaagtcgtgg ataagggggc ctctgcccag
     5941 tccttcatcg aaaggatgac taactttgat aaaaatctgc ctaacgaaaa ggtgcttcct
     6001 aaacactctc tgctgtacga gtacttcaca gtttataacg agctcaccaa ggtcaaatac
     6061 gtcacagaag ggatgagaaa gccagcattc ctgtctggag agcagaagaa agctatcgtg
     6121 gacctcctct tcaagacgaa ccggaaagtt accgtgaaac agctcaaaga agactatttc
     6181 aaaaagattg aatgtttcga ctctgttgaa atcagcggag tggaggatcg cttcaacgca
     6241 tccctgggaa cgtatcacga tctcctgaaa atcattaaag acaaggactt cctggacaat
     6301 gaggagaacg aggacattct tgaggacatt gtcctcaccc ttacgttgtt tgaagatagg
     6361 gagatgattg aagaacgctt gaaaacttac gctcatctct tcgacgacaa agtcatgaaa
     6421 cagctcaaga ggcgccgata tacaggatgg gggcggctgt caagaaaact gatcaatggg
     6481 atccgagaca agcagagtgg aaagacaatc ctggattttc ttaagtccga tggatttgcc
     6541 aaccggaact tcatgcagtt gatccatgat gactctctca cctttaagga ggacatccag
     6601 aaagcacaag tttctggcca gggggacagt cttcacgagc acatcgctaa tcttgcaggt
     6661 agcccagcta tcaaaaaggg aatactgcag accgttaagg tcgtggatga actcgtcaaa
     6721 gtaatgggaa ggcataagcc cgagaatatc gttatcgaga tggcccgaga gaaccaaact
     6781 acccagaagg gacagaagaa cagtagggaa aggatgaaga ggattgaaga gggtataaaa
     6841 gaactggggt cccaaatcct taaggaacac ccagttgaaa acacccagct tcagaatgag
     6901 aagctctacc tgtactacct gcagaacggc agggacatgt acgtggatca ggaactggac
     6961 atcaatcggc tctccgacta cgacgtggat gccatcgtgc cccagtcttt tctcaaagat
     7021 gattctattg ataataaagt gttgacaaga tccgataaaa atagagggaa gagtgataac
     7081 gtcccctcag aagaagttgt caagaaaatg aaaaattatt ggcggcagct gctgaacgcc
     7141 aaactgatca cacaacggaa gttcgataat ctgactaagg ctgaacgagg tggcctgtct
     7201 gagttggata aagccggctt catcaaaagg cagcttgttg agacacgcca gatcaccaag
     7261 cacgtggccc aaattctcga ttcacgcatg aacaccaagt acgatgaaaa tgacaaactg
     7321 attcgagagg tgaaagttat tactctgaag tctaagctgg tctcagattt cagaaaggac
     7381 tttcagtttt ataaggtgag agagatcaac aattaccacc atgcgcatga tgcctacctg
     7441 aatgcagtgg taggcactgc acttatcaaa aaatatccca agcttgaatc tgaatttgtt
     7501 tacggagact ataaagtgta cgatgttagg aaaatgatcg caaagtctga gcaggaaata
     7561 ggcaaggcca ccgctaagta cttcttttac agcaatatta tgaatttttt caagaccgag
     7621 attacactgg ccaatggaga gattcggaag cgaccactta tcgaaacaaa cggagaaaca
     7681 ggagaaatcg tgtgggacaa gggtagggat ttcgcgacag tccggaaggt cctgtccatg
     7741 ccgcaggtga acatcgttaa aaagaccgaa gtacagaccg gaggcttctc caaggaaagt
     7801 atcctcccga aaaggaacag cgacaagctg atcgcacgca aaaaagattg ggaccccaag
     7861 aaatacggcg gattcgattc tcctacagtc gcttacagtg tactggttgt ggccaaagtg
     7921 gagaaaggga agtctaaaaa actcaaaagc gtcaaggaac tgctgggcat cacaatcatg
     7981 gagcgatcaa gcttcgaaaa aaaccccatc gactttctcg aggcgaaagg atataaagag
     8041 gtcaaaaaag acctcatcat taagcttccc aagtactctc tctttgagct tgaaaacggc
     8101 cggaaacgaa tgctcgctag tgcgggcgag ctgcagaaag gtaacgagct ggcactgccc
     8161 tctaaatacg ttaatttctt gtatctggcc agccactatg aaaagctcaa agggtctccc
     8221 gaagataatg agcagaagca gctgttcgtg gaacaacaca aacactacct tgatgagatc
     8281 atcgagcaaa taagcgaatt ctccaaaaga gtgatcctcg ccgacgctaa cctcgataag
     8341 gtgctttctg cttacaataa gcacagggat aagcccatca gggagcaggc agaaaacatt
     8401 atccacttgt ttactctgac caacttgggc gcgcctgcag ccttcaagta cttcgacacc
     8461 accatagaca gaaagcggta cacctctaca aaggaggtcc tggacgccac actgattcat
     8521 cagtcaatta cggggctcta tgaaacaaga atcgacctct ctcagctcgg tggagacagc
     8581 agggctgacc ccaagaagaa gaggaaggtg gctagccgcg ccgacgcgct ggacgatttc
     8641 gatctcgaca tgctgggttc tgatgccctc gatgactttg acctggatat gttgggaagc
     8701 gacgcattgg atgactttga tctggacatg ctcggctccg atgctctgga cgatttcgat
     8761 ctcgatatgt taatcgctag cgagggcaga ggaagtcttc taacatgcgg tgacgtggag
     8821 gagaatcccg gccctggtac cgtgagcaag ggcgaggagc tgttcaccgg ggtggtgccc
     8881 atcctggtcg agctggacgg cgacgtaaac ggccacaagt tcagcgtgtc cggcgagggc
     8941 gagggcgatg ccacctacgg caagctgacc ctgaagttca tctgcaccac cggcaagctg
     9001 cccgtgccct ggcccaccct cgtgaccacc ctgacctacg gcgtgcagtg cttcagccgc
     9061 taccccgacc acatgaagca gcacgacttc ttcaagtccg ccatgcccga aggctacgtc
     9121 caggagcgca ccatcttctt caaggacgac ggcaactaca agacccgcgc cgaggtgaag
     9181 ttcgagggcg acaccctggt gaaccgcatc gagctgaagg gcatcgactt caaggaggac
     9241 ggcaacatcc tggggcacaa gctggagtac aactacaaca gccacaacgt ctatatcatg
     9301 gccgacaagc agaagaacgg catcaaggtg aacttcaaga tccgccacaa catcgaggac
     9361 ggcagcgtgc agctcgccga ccactaccag cagaacaccc ccatcggcga cggccccgtg
     9421 ctgctgcccg acaaccacta cctgagcacc cagtccgccc tgagcaaaga ccccaacgag
     9481 aagcgcgatc acatggtcct gctggagttc gtgaccgccg ccgggatcac tctcggcatg
     9541 gacgagctgt acaagaccgg ttgataatag ataacttcgt atagcataca ttatacgaag
     9601 ttatgaattc gatatcaagc ttatcgataa tcaacctctg gattacaaaa tttgtgaaag
     9661 attgactggt attcttaact atgttgctcc ttttacgcta tgtggatacg ctgctttaat
     9721 gcctttgtat catgctattg cttcccgtat ggctttcatt ttctcctcct tgtataaatc
     9781 ctggttgctg tctctttatg aggagttgtg gcccgttgtc aggcaacgtg gcgtggtgtg
     9841 cactgtgttt gctgacgcaa cccccactgg ttggggcatt gccaccacct gtcagctcct
     9901 ttccgggact ttcgctttcc ccctccctat tgccacggcg gaactcatcg ccgcctgcct
     9961 tgcccgctgc tggacagggg ctcggctgtt gggcactgac aattccgtgg tgttgtcggg
    10021 gaaatcatcg tcctttcctt ggctgctcgc ctgtgttgcc acctggattc tgcgcgggac
    10081 gtccttctgc tacgtccctt cggccctcaa tccagcggac cttccttccc gcggcctgct
    10141 gccggctctg cggcctcttc cgcgtcttcg ccttcgccct cagacgagtc ggatctccct
    10201 ttgggccgcc tccccgcatc gataccgtcg acctcgagac ctagaaaaac atggagcaat
    10261 cacaagtagc aatacagcag ctaccaatgc tgattgtgcc tggctagaag cacaagagga
    10321 ggaggaggtg ggttttccag tcacacctca ggtaccttta agaccaatga cttacaaggc
    10381 agctgtagat cttagccact ttttaaaaga aaagggggga ctggaagggc taattcactc
    10441 ccaacgaaga caagatatcc ttgatctgtg gatctaccac acacaaggct acttccctga
    10501 ttggcagaac tacacaccag ggccagggat cagatatcca ctgacctttg gatggtgcta
    10561 caagctagta ccagttgagc aagagaaggt agaagaagcc aatgaaggag agaacacccg
    10621 cttgttacac cctgtgagcc tgcatgggat ggatgacccg gagagagaag tattagagtg
    10681 gaggtttgac agccgcctag catttcatca catggcccga gagctgcatc cggactgtac
    10741 tgggtctctc tggttagacc agatctgagc ctgggagctc tctggctaac tagggaaccc
    10801 actgcttaag cctcaataaa gcttgccttg agtgcttcaa gtagtgtgtg cccgtctgtt
    10861 gtgtgactct ggtaactaga gatccctcag acccttttag tcagtgtgga aaatctctag
    10921 cagggcccgt ttaaacccgc tgatcagcct cgactgtgcc ttctagttgc cagccatctg
    10981 ttgtttgccc ctcccccgtg ccttccttga ccctggaagg tgccactccc actgtccttt
    11041 cctaataaaa tgaggaaatt gcatcgcatt gtctgagtag gtgtcattct attctggggg
    11101 gtggggtggg gcaggacagc aagggggagg attgggaaga caatagcagg catgctgggg
    11161 atgcggtggg ctctatggct tctgaggcgg aaagaaccag ctggggctct agggggtatc
    11221 cccacgcgcc ctgtagcggc gcattaagcg cggcgggtgt ggtggttacg cgcagcgtga
    11281 ccgctacact tgccagcgcc ctagcgcccg ctcctttcgc tttcttccct tcctttctcg
    11341 ccacgttcgc cggctttccc cgtcaagctc taaatcgggg gctcccttta gggttccgat
    11401 ttagtgcttt acggcacctc gaccccaaaa aacttgatta gggtgatggt tcacgtagtg
    11461 ggccatcgcc ctgatagacg gtttttcgcc ctttgacgtt ggagtccacg ttctttaata
    11521 gtggactctt gttccaaact ggaacaacac tcaaccctat ctcggtctat tcttttgatt
    11581 tataagggat tttgccgatt tcggcctatt ggttaaaaaa tgagctgatt taacaaaaat
    11641 ttaacgcgaa ttaattctgt ggaatgtgtg tcagttaggg tgtggaaagt ccccaggctc
    11701 cccagcaggc agaagtatgc aaagcatgca tctcaattag tcagcaacca ggtgtggaaa
    11761 gtccccaggc tccccagcag gcagaagtat gcaaagcatg catctcaatt agtcagcaac
    11821 catagtcccg cccctaactc cgcccatccc gcccctaact ccgcccagtt ccgcccattc
    11881 tccgccccat ggctgactaa ttttttttat ttatgcagag gccgaggccg cctctgcctc
    11941 tgagctattc cagaagtagt gaggaggctt ttttggaggc ctaggctttt gcaaaaagct
    12001 cccgggagct tgtatatcca ttttcggatc tgatcagcac gtgttgacaa ttaatcatcg
    12061 gcatagtata tcggcatagt ataatacgac aaggtgagga actaaaccat ggccaagttg
    12121 accagtgccg ttccggtgct caccgcgcgc gacgtcgccg gagcggtcga gttctggacc
    12181 gaccggctcg ggttctcccg ggacttcgtg gaggacgact tcgccggtgt ggtccgggac
    12241 gacgtgaccc tgttcatcag cgcggtccag gaccaggtgg tgccggacaa caccctggcc
    12301 tgggtgtggg tgcgcggcct ggacgagctg tacgccgagt ggtcggaggt cgtgtccacg
    12361 aacttccggg acgcctccgg gccggccatg accgagatcg gcgagcagcc gtgggggcgg
    12421 gagttcgccc tgcgcgaccc ggccggcaac tgcgtgcact tcgtggccga ggagcaggac
    12481 tgacacgtgc tacgagattt cgattccacc gccgccttct atgaaaggtt gggcttcgga
    12541 atcgttttcc gggacgccgg ctggatgatc ctccagcgcg gggatctcat gctggagttc
    12601 ttcgcccacc ccaacttgtt tattgcagct tataatggtt acaaataaag caatagcatc
    12661 acaaatttca caaataaagc atttttttca ctgcattcta gttgtggttt gtccaaactc
    12721 atcaatgtat cttatcatgt ctgtataccg tcgacctcta gctagagctt ggcgtaatca
    12781 tggtcatagc tgtttcctgt gtgaaattgt tatccgctca caattccaca caacatacga
    12841 gccggaagca taaagtgtaa agcctggggt gcctaatgag tgagctaact cacattaatt
    12901 gcgttgcgct cactgcccgc tttccagtcg ggaaacctgt cgtgccagct gcattaatga
    12961 atcggccaac gcgcggggag aggcggtttg cgtattgggc gctcttccgc ttcctcgctc
    13021 actgactcgc tgcgctcggt cgttcggctg cggcgagcgg tatcagctca ctcaaaggcg
    13081 gtaatacggt tatccacaga atcaggggat aacgcaggaa agaacatgtg agcaaaaggc
    13141 cagcaaaagg ccaggaaccg taaaaaggcc gcgttgctgg cgtttttcca taggctccgc
    13201 ccccctgacg agcatcacaa aaatcgacgc tcaagtcaga ggtggcgaaa cccgacagga
    13261 ctataaagat accaggcgtt tccccctgga agctccctcg tgcgctctcc tgttccgacc
    13321 ctgccgctta ccggatacct gtccgccttt ctcccttcgg gaagcgtggc gctttctcat
    13381 agctcacgct gtaggtatct cagttcggtg taggtcgttc gctccaagct gggctgtgtg
    13441 cacgaacccc ccgttcagcc cgaccgctgc gccttatccg gtaactatcg tcttgagtcc
    13501 aacccggtaa gacacgactt atcgccactg gcagcagcca ctggtaacag gattagcaga
    13561 gcgaggtatg taggcggtgc tacagagttc ttgaagtggt ggcctaacta cggctacact
    13621 agaagaacag tatttggtat ctgcgctctg ctgaagccag ttaccttcgg aaaaagagtt
    13681 ggtagctctt gatccggcaa acaaaccacc gctggtagcg gtggtttttt tgtttgcaag
    13741 cagcagatta cgcgcagaaa aaaaggatct caagaagatc ctttgatctt ttctacgggg
    13801 tctgacgctc agtggaacga aaactcacgt taagggattt tggtcatgag attatcaaaa
    13861 aggatcttca cctagatcct tttaaattaa aaatgaagtt ttaaatcaat ctaaagtata
    13921 tatgagtaaa cttggtctga cagttaccaa tgcttaatca gtgaggcacc tatctcagcg
    13981 atctgtctat ttcgttcatc catagttgcc tgactccccg tcgtgtagat aactacgata
    14041 cgggagggct taccatctgg ccccagtgct gcaatgatac cgcgagaccc acgctcaccg
    14101 gctccagatt tatcagcaat aaaccagcca gccggaaggg ccgagcgcag aagtggtcct
    14161 gcaactttat ccgcctccat ccagtctatt aattgttgcc gggaagctag agtaagtagt
    14221 tcgccagtta atagtttgcg caacgttgtt gccattgcta caggcatcgt ggtgtcacgc
    14281 tcgtcgtttg gtatggcttc attcagctcc ggttcccaac gatcaaggcg agttacatga
    14341 tcccccatgt tgtgcaaaaa agcggttagc tccttcggtc ctccgatcgt tgtcagaagt
    14401 aagttggccg cagtgttatc actcatggtt atggcagcac tgcataattc tcttactgtc
    14461 atgccatccg taagatgctt ttctgtgact ggtgagtact caaccaagtc attctgagaa
    14521 tagtgtatgc ggcgaccgag ttgctcttgc ccggcgtcaa tacgggataa taccgcgcca
    14581 catagcagaa ctttaaaagt gctcatcatt ggaaaacgtt cttcggggcg aaaactctca
    14641 aggatcttac cgctgttgag atccagttcg atgtaaccca ctcgtgcacc caactgatct
    14701 tcagcatctt ttactttcac cagcgtttct gggtgagcaa aaacaggaag gcaaaatgcc
    14761 gcaaaaaagg gaataagggc gacacggaaa tgttgaatac tcatactctt cctttttcaa
    14821 tattattgaa gcatttatca gggttattgt ctcatgagcg gatacatatt tgaatgtatt
    14881 tagaaaaata aacaaatagg ggttccgcgc acatttcccc gaaaagtgcc acctgac
//
