pXPR_120

LOCUS       pXPR_120               15513 bp ds-DNA     circular SYN 06-AUG-2018
DEFINITION  for CRISPRa, lentiviral expression of dCas9-VPR 2A BlastR.
ACCESSION   .
VERSION     .
KEYWORDS    pXPR_120
SOURCE      synthetic DNA construct
  ORGANISM  synthetic DNA construct
REFERENCE   1  (bases 1 to 15513)
  AUTHORS   Najm FJ, Strand C, Donovan KF, Hegde M, Sanson KR, Vaimberg EW, 
            Sullender ME, Hartenian E, Kalani Z, Fusi N, Listgarten J, Younger 
            ST, Bernstein BE, Root DE, Doench JG
  TITLE     Orthologous CRISPR-Cas9 enzymes for combinatorial genetic screens.
  JOURNAL   Nat Biotechnol. 2018 Feb;36(2):179-189. doi: 10.1038/nbt.4048. Epub 
            2017 Dec 18.
  PUBMED    29251726
REFERENCE   2  (bases 1 to 15513)
  AUTHORS   .
  TITLE     Direct Submission
  JOURNAL   Exported Aug 6, 2018 from SnapGene Server 1.1.58
            http://www.snapgene.com
FEATURES             Location/Qualifiers
     source          1..15513
                     /organism="synthetic DNA construct"
                     /mol_type="other DNA"
     primer_bind     complement(30..49)
                     /label=SV40pro-F
                     /note="SV40 promoter/origin, forward primer"
     primer_bind     276..296
                     /label=pBABE 3'
                     /note="SV40 enhancer, reverse primer for pBABE vectors"
     rep_origin      complement(305..733)
                     /direction=LEFT
                     /label=f1 ori
                     /note="f1 bacteriophage origin of replication; arrow 
                     indicates direction of (+) strand synthesis"
     primer_bind     complement(415..436)
                     /label=F1ori-F
                     /note="F1 origin, forward primer"
     primer_bind     627..646
                     /label=F1ori-R
                     /note="F1 origin, reverse primer"
     polyA_signal    779..1003
                     /label=bGH poly(A) signal
                     /note="bovine growth hormone polyadenylation signal"
     primer_bind     992..1009
                     /label=BGH-rev
                     /note="Bovine growth hormone terminator, reverse primer. 
                     Also called BGH reverse"
     LTR             1035..1215
                     /label=5' LTR (truncated)
                     /note="truncated 5' long terminal repeat (LTR) from HIV-1"
     primer_bind     1720..1736
                     /label=pBluescriptKS
                     /note="For pBluescript vector"
     primer_bind     1721..1737
                     /label=KS primer
                     /note="common sequencing primer, one of multiple similar 
                     variants"
     misc_feature    1740..2328
                     /label=WPRE
                     /note="woodchuck hepatitis virus posttranscriptional 
                     regulatory element"
     CDS             1846..1857
                     /codon_start=1
                     /product="Factor Xa recognition and cleavage site"
                     /label=Factor Xa site
                     /translation="IEGR"
     primer_bind     2255..2275
                     /label=WPRE-R
                     /note="WPRE, reverse primer"
     CDS             complement(2353..2751)
                     /codon_start=1
                     /gene="Aspergillus terreus BSD"
                     /product="blasticidin S deaminase"
                     /label=BSD
                     /note="confers resistance to blasticidin"
                     /translation="MAKPLSQEESTLIERATATINSIPISEDYSVASAALSSDGRIFTG
                     VNVYHFTGGPCAELVVLGTAAAAAAGNLTCIVAIGNENRGILSPCGRCRQVLLDLHPGI
                     KAIVKDSDGQPTAVGIRELLPSGYVWEG"
     CDS             complement(2752..2805)
                     /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             complement(2821..3390)
                     /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             complement(3409..3765)
                     /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             complement(4195..4215)
                     /codon_start=1
                     /product="nuclear localization signal of SV40 (simian virus
                     40) large T antigen"
                     /label=SV40 NLS
                     /translation="PKKKRKV"
     CDS             complement(4240..4389)
                     /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             complement(4429..4449)
                     /codon_start=1
                     /product="nuclear localization signal of SV40 (simian virus
                     40) large T antigen"
                     /label=SV40 NLS
                     /translation="PKKKRKV"
     CDS             complement(4504..8604)
                     /codon_start=1
                     /product="catalytically dead mutant of the Cas9 
                     endonuclease from the Streptococcus pyogenes Type II 
                     CRISPR/Cas system"
                     /label=dCas9
                     /note="RNA-guided DNA-binding protein that lacks 
                     endonuclease activity due to the D10A mutation in the RuvC 
                     catalytic domain and the N863A mutation in the HNH 
                     catalytic domain"
                     /translation="DKKYSIGLAIGTNSVGWAVITDEYKVPSKKFKVLGNTDRHSIKKN
                     LIGALLFDSGETAEATRLKRTARRRYTRRKNRICYLQEIFSNEMAKVDDSFFHRLEESF
                     LVEEDKKHERHPIFGNIVDEVAYHEKYPTIYHLRKKLVDSTDKADLRLIYLALAHMIKF
                     RGHFLIEGDLNPDNSDVDKLFIQLVQTYNQLFEENPINASGVDAKAILSARLSKSRRLE
                     NLIAQLPGEKKNGLFGNLIALSLGLTPNFKSNFDLAEDAKLQLSKDTYDDDLDNLLAQI
                     GDQYADLFLAAKNLSDAILLSDILRVNTEITKAPLSASMIKRYDEHHQDLTLLKALVRQ
                     QLPEKYKEIFFDQSKNGYAGYIDGGASQEEFYKFIKPILEKMDGTEELLVKLNREDLLR
                     KQRTFDNGSIPHQIHLGELHAILRRQEDFYPFLKDNREKIEKILTFRIPYYVGPLARGN
                     SRFAWMTRKSEETITPWNFEEVVDKGASAQSFIERMTNFDKNLPNEKVLPKHSLLYEYF
                     TVYNELTKVKYVTEGMRKPAFLSGEQKKAIVDLLFKTNRKVTVKQLKEDYFKKIECFDS
                     VEISGVEDRFNASLGTYHDLLKIIKDKDFLDNEENEDILEDIVLTLTLFEDREMIEERL
                     KTYAHLFDDKVMKQLKRRRYTGWGRLSRKLINGIRDKQSGKTILDFLKSDGFANRNFMQ
                     LIHDDSLTFKEDIQKAQVSGQGDSLHEHIANLAGSPAIKKGILQTVKVVDELVKVMGRH
                     KPENIVIEMARENQTTQKGQKNSRERMKRIEEGIKELGSQILKEHPVENTQLQNEKLYL
                     YYLQNGRDMYVDQELDINRLSDYDVDHIVPQSFLKDDSIDNKVLTRSDKARGKSDNVPS
                     EEVVKKMKNYWRQLLNAKLITQRKFDNLTKAERGGLSELDKAGFIKRQLVETRQITKHV
                     AQILDSRMNTKYDENDKLIREVKVITLKSKLVSDFRKDFQFYKVREINNYHHAHDAYLN
                     AVVGTALIKKYPKLESEFVYGDYKVYDVRKMIAKSEQEIGKATAKYFFYSNIMNFFKTE
                     ITLANGEIRKRPLIETNGETGEIVWDKGRDFATVRKVLSMPQVNIVKKTEVQTGGFSKE
                     SILPKRNSDKLIARKKDWDPKKYGGFDSPTVAYSVLVVAKVEKGKSKKLKSVKELLGIT
                     IMERSSFEKNPIDFLEAKGYKEVKKDLIIKLPKYSLFELENGRKRMLASAGELQKGNEL
                     ALPSKYVNFLYLASHYEKLKGSPEDNEQKQLFVEQHKHYLDEIIEQISEFSKRVILADA
                     NLDKVLSAYNKHRDKPIREQAENIIHLFTLTNLGAPAAFKYFDTTIDRKRYTSTKEVLD
                     ATLIHQSITGLYETRIDLSQLGGD"
     CDS             complement(8605..8652)
                     /codon_start=1
                     /product="bipartite nuclear localization signal from 
                     nucleoplasmin"
                     /label=nucleoplasmin NLS
                     /translation="KRPAATKKAGQAKKKK"
     promoter        complement(8668..9846)
                     /label=EF-1-alpha promoter
                     /note="strong constitutive promoter for human elongation 
                     factor EF-1-alpha"
     intron          8677..9615
                     /label=EF-1-alpha intron A
                     /note="intron upstream of the start codon of human 
                     EF-1-alpha"
     primer_bind     complement(8700..8720)
                     /label=EF1a-F
                     /note="Human elongation factor-1a promoter, forward primer"
     misc_feature    9976..10093
                     /label=cPPT/CTS
                     /note="central polypurine tract and central termination 
                     sequence of HIV-1"
     misc_feature    10620..10853
                     /label=RRE
                     /note="The Rev response element (RRE) of HIV-1 allows for 
                     Rev-dependent mRNA export from the nucleus to the 
                     cytoplasm."
     misc_feature    11346..11471
                     /label=HIV-1 Psi
                     /note="packaging signal of human immunodeficiency virus 
                     type 1"
     LTR             11518..11698
                     /label=5' LTR (truncated)
                     /note="truncated 5' long terminal repeat (LTR) from HIV-1"
     promoter        complement(11716..11914)
                     /label=CMV promoter
                     /note="human cytomegalovirus (CMV) immediate early 
                     promoter"
     primer_bind     complement(11741..11761)
                     /label=CMV-F
                     /note="Human CMV immediate early promoter, forward primer"
     enhancer        11916..12295
                     /label=CMV enhancer
                     /note="human cytomegalovirus immediate early enhancer"
     primer_bind     12467..12486
                     /label=pRS-marker
                     /note="pRS vectors, use to sequence yeast selectable 
                     marker"
     promoter        12561..12665
                     /gene="bla"
                     /label=AmpR promoter
     CDS             12666..13526
                     /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     complement(12884..12903)
                     /label=Amp-R
                     /note="Ampicillin resistance gene, reverse primer"
     rep_origin      13697..14285
                     /direction=RIGHT
                     /label=ori
                     /note="high-copy-number ColE1/pMB1/pBR322/pUC origin of 
                     replication"
     primer_bind     14186..14205
                     /label=pBR322ori-F
                     /note="pBR322 origin, forward primer"
     primer_bind     14439..14456
                     /label=L4440
                     /note="L4440 vector, forward primer"
     protein_bind    14573..14594
                     /label=CAP binding site
                     /bound_moiety="E. coli catabolite activator protein"
                     /note="CAP binding activates transcription in the presence 
                     of cAMP."
     promoter        14609..14639
                     /label=lac promoter
                     /note="promoter for the E. coli lac operon"
     protein_bind    14647..14663
                     /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)."
     primer_bind     14652..14674
                     /label=M13/pUC Reverse
                     /note="In lacZ gene"
     primer_bind     14671..14687
                     /label=M13 rev
                     /note="common sequencing primer, one of multiple similar 
                     variants"
     primer_bind     14671..14687
                     /label=M13 Reverse
                     /note="In lacZ gene. Also called M13-rev"
     polyA_signal    14736..14857
                     /label=SV40 poly(A) signal
                     /note="SV40 polyadenylation signal"
     primer_bind     complement(14747..14766)
                     /label=EBV-rev
                     /note="SV40 polyA terminator, reverse primer"
     primer_bind     14801..14820
                     /label=SV40pA-R
                     /note="SV40 polyA, reverse primer"
     CDS             complement(14987..15361)
                     /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"
     promoter        complement(15380..15427)
                     /label=EM7 promoter
                     /note="synthetic bacterial promoter "
     promoter        complement(15475..291)
                     /label=SV40 promoter
                     /note="SV40 enhancer and early promoter"
     rep_origin      15489..111
                     /label=SV40 ori
                     /note="SV40 origin of replication"
ORIGIN
        1 cttctggaat agctcagagg cagaggcggc ctcggcctct gcataaataa aaaaaattag
       61 tcagccatgg ggcggagaat gggcggaact gggcggagtt aggggcggga tgggcggagt
      121 taggggcggg actatggttg ctgactaatt gagatgcatg ctttgcatac ttctgcctgc
      181 tggggagcct ggggactttc cacacctggt tgctgactaa ttgagatgca tgctttgcat
      241 acttctgcct gctggggagc ctggggactt tccacaccct aactgacaca cattccacag
      301 aattaattcg cgttaaattt ttgttaaatc agctcatttt ttaaccaata ggccgaaatc
      361 ggcaaaatcc cttataaatc aaaagaatag accgagatag ggttgagtgt tgttccagtt
      421 tggaacaaga gtccactatt aaagaacgtg gactccaacg tcaaagggcg aaaaaccgtc
      481 tatcagggcg atggcccact acgtgaacca tcaccctaat caagtttttt ggggtcgagg
      541 tgccgtaaag cactaaatcg gaaccctaaa gggagccccc gatttagagc ttgacgggga
      601 aagccggcga acgtggcgag aaaggaaggg aagaaagcga aaggagcggg cgctagggcg
      661 ctggcaagtg tagcggtcac gctgcgcgta accaccacac ccgccgcgct taatgcgccg
      721 ctacagggcg cgtggggata ccccctagag ccccagctgg ttctttccgc ctcagaagcc
      781 atagagccca ccgcatcccc agcatgcctg ctattgtctt cccaatcctc ccccttgctg
      841 tcctgcccca ccccaccccc cagaatagaa tgacacctac tcagacaatg cgatgcaatt
      901 tcctcatttt attaggaaag gacagtggga gtggcacctt ccagggtcaa ggaaggcacg
      961 ggggaggggc aaacaacaga tggctggcaa ctagaaggca cagtcgaggc tgatcagcgg
     1021 gtttaaacgg gccctgctag agattttcca cactgactaa aagggtctga gggatctcta
     1081 gttaccagag tcacacaaca gacgggcaca cactacttga agcactcaag gcaagcttta
     1141 ttgaggctta agcagtgggt tccctagtta gccagagagc tcccaggctc agatctggtc
     1201 taaccagaga gacccagtac agtccggatg cagctctcgg gccatgtgat gaaatgctag
     1261 gcggctgtca aacctccact ctaatacttc tctctccggg tcatccatcc catgcaggct
     1321 cacagggtgt aacaagcggg tgttctctcc ttcattggct tcttctacct tctcttgctc
     1381 aactggtact agcttgtagc accatccaaa ggtcagtgga tatctgatcc ctggccctgg
     1441 tgtgtagttc tgccaatcag ggaagtagcc ttgtgtgtgg tagatccaca gatcaaggat
     1501 atcttgtctt cgttgggagt gaattagccc ttccagtccc cccttttctt ttaaaaagtg
     1561 gctaagatct acagctgcct tgtaagtcat tggtcttaaa ggtacctgag gtgtgactgg
     1621 aaaacccacc tcctcctcct cttgtgcttc tagccaggca caatcagcat tggtagctgc
     1681 tgtattgcta cttgtgattg ctccatgttt ttctaggtct cgaggtcgac ggtatcgatg
     1741 cggggaggcg gcccaaaggg agatccgact cgtctgaggg cgaaggcgaa gacgcggaag
     1801 aggccgcaga gccggcagca ggccgcggga aggaaggtcc gctggattga gggccgaagg
     1861 gacgtagcag aaggacgtcc cgcgcagaat ccaggtggca acacaggcga gcagccaagg
     1921 aaaggacgat gatttccccg acaacaccac ggaattgtca gtgcccaaca gccgagcccc
     1981 tgtccagcag cgggcaaggc aggcggcgat gagttccgcc gtggcaatag ggagggggaa
     2041 agcgaaagtc ccggaaagga gctgacaggt ggtggcaatg ccccaaccag tgggggttgc
     2101 gtcagcaaac acagtgcaca ccacgccacg ttgcctgaca acgggccaca actcctcata
     2161 aagagacagc aaccaggatt tatacaagga ggagaaaatg aaagccatac gggaagcaat
     2221 agcatgatac aaaggcatta aagcagcgta tccacatagc gtaaaaggag caacatagtt
     2281 aagaatacca gtcaatcttt cacaaatttt gtaatccaga ggttgattac cgataagctt
     2341 gatatcgaat tcttagccct cccacacata accagagggc agcaattcac gaatcccaac
     2401 tgccgtcggc tgtccatcac tgtccttcac tatggctttg atcccaggat gcagatcgag
     2461 aagcacctgt cggcaccgtc cgcaggggct caagatgccc ctgttctcat ttccgatcgc
     2521 gacgatacaa gtcaggttgc cagctgccgc agcagcagca gtgcccagca ccacgagttc
     2581 tgcacaaggt cccccagtaa aatgatatac attgacacca gtgaagatgc ggccgtcgct
     2641 agagagagct gcgctggcga cgctgtagtc ttcagagatg gggatgctgt tgattgtagc
     2701 cgttgctctt tcaatgaggg tggattcttc ttgagacaaa ggcttggcca ttgggccagg
     2761 attctcctcg acgtcaccgc atgttagcag acttcctctg ccctctccac tgcctgtaca
     2821 gaagaggctg gtgtcgaaga tggacagccc ggtggagatg tgcatggcgt gcagcaggca
     2881 ctcgtcgttg aggaaggtgt ccaggatctc gttgagctca ggggtgagcg gcgagtccaa
     2941 gttgaggtcc tcggtcatgc tctcgagggt ggtggtgagc tcgtccaggt ggccgcgggg
     3001 cggagggtgg gacaggtcca tctggccgca gatggcagcc tcctccttct gggggatgac
     3061 ggtgtcggcc atctcgcgca gggccttgac ggcctgggag gtctcctcgt cagggtcctc
     3121 cagcaggtgg gaggcctcgg gcgtgacagc aggagcaggg tccagcggct ggggcacagg
     3181 cgcaggggtg aggctgccaa ctggctcgtg cacggggcca gttggggttg gtgccaacga
     3241 ggcaggcagc gggcggttgg cccagggtga gccaggaggg tggaaggggc ggatccgctt
     3301 gggctggcag acctcacgcc cctcgaagac gtccgagatg gcagagccag cctccggctt
     3361 gggcaggaac atgccctcgc gggagtcgcg ggagccggag ccagagccca gcagggcaga
     3421 gaagtccatg tccgcgatgg aggagaagtc ttcgtcgccg ctcagcaggc cgttgggcag
     3481 gcctggagcc cccaggggag caggggcagg gtcaggaggg cgttgggcac cggtcaccag
     3541 gcgggtgatg gcctctgggt actccatgag catgggctcg gtggtgtggg gggccacggg
     3601 gatgccctgg ttgagcagct gctggaactc ggagttgtcc accgatgcca agtcggtgaa
     3661 gacagcaggg tcggtggagt tgcccagcag ggcccccagg tcctcgtcgt cgaactgaag
     3721 ttggagcagc gcctcggaca gggtgccctc gcctgcctgg gtaggcttgg gcgcaggagg
     3781 cgccacggcc tgcggagggc caggtgccaa cacaggcacc ggggcagggg cctgggccag
     3841 ggcagagacc atggcaggag caggagcagg agcaggagcc tggggcagca cctgcggagg
     3901 agcaggtgcc aacgcagagg cctgggagat ctggccagag gggaagacca tggtggggaa
     3961 ctcgtcgtag ttgatggtgg acagcgagga ggtgaagggg tagggctggg gagcaggctt
     4021 gggcacagag gcagaggagc ggctgggcac cgcgatgcgc cgcggaggcg gcctcgggtc
     4081 agttgggcca gagaagggcg acttcttcat gatggacttg aaagtttcgt aggtgcgctt
     4141 gcgcttttcc tcgatacggt ggcggtcgtc ggtgtcgggc aggtactggc tgccaacttt
     4201 gcgcttcttc ttgggagacc cagaggagcg ggagttgatg agcatgtcca ggtcgaagtc
     4261 gtcgagggcg tccgagccca gcatgtccag gtcgaagtcg tccagcgcgt cgctgcccag
     4321 catgtccagg tcgaagtcgt ccagggcgtc ggagcccagc atgtccaggt cgaagtcgtc
     4381 cagggcgtcc gcgcggccgg agccagaggc ctcggatcca gcggccgcca ccttcctctt
     4441 tttcttaggt ccgctacctc ctcctccgct tcctcctcct ccgcttccac ctcctccagc
     4501 gctgtcgcct cccagctgag acaggtcgat ccgtgtctcg tacaggccgg tgatgctctg
     4561 gtggatcagg gtggcgtcca gcacctcttt ggtgctggtg tacctcttcc ggtcgatggt
     4621 ggtgtcaaag tacttgaagg cggcaggggc tcccagattg gtcagggtaa acaggtggat
     4681 gatattctcg gcctgctctc tgatgggctt atcccggtgc ttgttgtagg cggacagcac
     4741 tttgtccaga ttagcgtcgg ccaggatcac tctcttggag aactcgctga tctgctcgat
     4801 gatctcgtcc aggtagtgct tgtgctgttc cacaaacagc tgtttctgct cattatcctc
     4861 gggggagccc ttcagcttct catagtggct ggccaggtac aggaagttca catatttgga
     4921 gggcagggcc agttcgtttc ccttctgcag ttcgccggca gaggccagca ttctcttccg
     4981 gccgttttcc agctcgaaca gggagtactt aggcagcttg atgatcaggt cctttttcac
     5041 ttctttgtag cccttggctt ccagaaagtc gatgggattc ttctcgaagc tgcttctttc
     5101 catgatggtg atccccagca gctctttcac actcttcagt ttcttggact tgcccttttc
     5161 cactttggcc accaccagca cagaataggc cacggtgggg ctgtcgaagc cgccgtactt
     5221 cttagggtcc cagtccttct ttctggcgat cagcttatcg ctgttcctct tgggcaggat
     5281 agactctttg ctgaagccgc ctgtctgcac ctcggtcttt ttcacgatat tcacttgggg
     5341 catgctcagc actttccgca cggtggcaaa atcccggccc ttatcccaca cgatctcccc
     5401 ggtttcgccg tttgtctcga tcagaggccg cttccggatc tcgccgttgg ccagggtaat
     5461 ctcggtcttg aaaaagttca tgatgttgct gtagaagaag tacttggcgg tagccttgcc
     5521 gatttcctgc tcgctcttgg cgatcatctt ccgcacgtcg tacaccttgt agtcgccgta
     5581 cacgaactcg ctttccagct tagggtactt tttgatcagg gcggttccca cgacggcgtt
     5641 caggtaggcg tcgtgggcgt ggtggtagtt gttgatctcg cgcactttgt aaaactggaa
     5701 atccttccgg aaatcggaca ccagcttgga cttcagggtg atcactttca cttcccggat
     5761 cagcttgtca ttctcgtcgt acttagtgtt catccgggag tccaggatct gtgccacgtg
     5821 ctttgtgatc tgccgggttt ccaccagctg tctcttgatg aagccggcct tatccagttc
     5881 gctcaggccg cctctctcgg ccttggtcag attgtcgaac tttctctggg taatcagctt
     5941 ggcgttcagc agctgccgcc agtagttctt catcttcttc acgacctctt cggagggcac
     6001 gttgtcgctc ttgccccggg ccttgtcgct tctggtcagc accttgttgt cgatggagtc
     6061 gtccttcaga aagctctgag gcacgatgtg gtccacatcg tagtcggaca gccggttgat
     6121 gtccagttcc tggtccacgt acatatcccg cccattctgc aggtagtaca ggtacagctt
     6181 ctcgttctgc agctgggtgt tttccacggg gtgttctttc aggatctggc tgcccagctc
     6241 tttgatgccc tcttcgatcc gcttcattct ctcgcggctg ttcttctgtc ccttctgggt
     6301 ggtctggttc tctctggcca tttcgatcac gatgttctcg ggcttgtgcc ggcccatcac
     6361 tttcacgagc tcgtccacca ccttcactgt ctgcaggatg cccttcttaa tggcggggct
     6421 gccggccaga ttggcaatgt gctcgtgcag gctatcgccc tggccggaca cctgggcttt
     6481 ctggatgtcc tctttaaagg tcaggctgtc gtcgtggatc agctgcatga agtttctgtt
     6541 ggcgaagccg tcggacttca ggaaatccag gattgtcttg ccggactgct tgtcccggat
     6601 gccgttgatc agcttccggc tcagcctgcc ccagccggtg tatctccgcc gcttcagctg
     6661 cttcatcact ttgtcgtcga acaggtgggc ataggttttc agccgttcct cgatcatctc
     6721 tctgtcctca aacagtgtca gggtcagcac gatatcttcc agaatgtcct cgttttcctc
     6781 attgtccagg aagtccttgt ccttgataat tttcagcaga tcgtggtatg tgcccaggga
     6841 ggcgttgaac cgatcttcca cgccggagat ttccacggag tcgaagcact cgattttctt
     6901 gaagtagtcc tctttcagct gcttcacggt cactttccgg ttggtcttga acagcaggtc
     6961 cacgatggcc tttttctgct cgccgctcag gaaggcgggc tttctcattc cctcggtcac
     7021 gtatttcact ttggtcagct cgttatacac ggtgaagtac tcgtacagca ggctgtgctt
     7081 gggcagcacc ttctcgttgg gcaggttctt atcgaagttg gtcatccgct cgatgaagct
     7141 ctgggcggaa gcgcccttgt ccaccacttc ctcgaagttc cagggggtga tggtttcctc
     7201 gctctttctg gtcatccagg cgaatctgct gtttcccctg gccagagggc ccacgtagta
     7261 ggggatgcgg aaggtcagga tcttctcgat cttttcccgg ttgtccttca ggaatgggta
     7321 aaaatcttcc tgccgccgca gaatggcgtg cagctctccc aggtggatct ggtgggggat
     7381 gctgccgttg tcgaaggtcc gctgcttccg cagcaggtcc tctctgttca gcttcacgag
     7441 cagttcctcg gtgccgtcca tcttttccag gatgggcttg atgaacttgt agaactcttc
     7501 ctggctggct ccgccgtcaa tgtagccggc gtagccgttc ttgctctggt cgaagaaaat
     7561 ctctttgtac ttctcaggca gctgctgccg cacgagagct ttcagcaggg tcaggtcctg
     7621 gtggtgctcg tcgtatctct tgatcataga ggcgctcagg ggggccttgg tgatctcggt
     7681 gttcactctc aggatgtcgc tcagcaggat ggcgtcggac aggttcttgg cggccagaaa
     7741 caggtcggcg tactggtcgc cgatctgggc cagcaggttg tccaggtcgt cgtcgtaggt
     7801 gtccttgctc agctgcagtt tggcatcctc ggccaggtcg aagttgctct tgaagttggg
     7861 ggtcaggccc aggctcaggg caatcaggtt gccgaacagg ccattcttct tctcgccggg
     7921 cagctgggcg atcagatttt ccagccgtct gctcttgctc agtctggcag acaggatggc
     7981 cttggcgtcc acgccgctgg cgttgatggg gttttcctcg aacagctggt tgtaggtctg
     8041 caccagctgg atgaacagct tgtccacgtc gctgttgtcg gggttcaggt cgccctcgat
     8101 caggaagtgg ccccggaact tgatcatgtg ggccagggcc agatagatca gccgcaggtc
     8161 ggccttgtcg gtgctgtcca ccagtttctt tctcaggtgg tagatggtgg ggtacttctc
     8221 gtggtaggcc acctcgtcca cgatgttgcc gaagatgggg tgccgctcgt gcttcttatc
     8281 ctcttccacc aggaaggact cttccagtct gtggaagaag ctgtcgtcca ccttggccat
     8341 ctcgttgctg aagatctctt gcagatagca gatccggttc ttccgtctgg tgtatcttct
     8401 tctggcggtt ctcttcagcc gggtggcctc ggctgtttcg ccgctgtcga acagcagggc
     8461 tccgatcagg ttcttcttga tgctgtgccg gtcggtgttg cccagcacct tgaatttctt
     8521 gctgggcacc ttgtactcgt cggtgatcac ggcccagccc acagagttgg tgccgatggc
     8581 caggccgatg ctgtacttct tgtccttttt cttttttgcc tggccggcct ttttcgtggc
     8641 cgccggcctt ttcatggtgg ccgtacgtca cgacacctga aatggaagaa aaaaactttg
     8701 aaccactgtc tgaggcttga gaatgaacca agatccaaac tcaaaaaggg caaattccaa
     8761 ggagaattac atcaagtgcc aagctggcct aacttcagtc tccacccact cagtgtgggg
     8821 aaactccatc gcataaaacc cctcccccca acctaaagac gacgtactcc aaaagctcga
     8881 gaactaatcg aggtgcctgg acggcgcccg gtactccgtg gagtcacatg aagcgacggc
     8941 tgaggacgga aaggcccttt tcctttgtgt gggtgactca cccgcccgct ctcccgagcg
     9001 ccgcgtcctc cattttgagc tccctgcagc agggccggga agcggccatc tttccgctca
     9061 cgcaactggt gccgaccggg ccagccttgc cgcccagggc ggggcgatac acggcggcgc
     9121 gaggccaggc accagagcag gccggccagc ttgagactac ccccgtccga ttctcggtgg
     9181 ccgcgctcgc aggccccgcc tcgccgaaca tgtgcgctgg gacgcacggg ccccgtcgcc
     9241 gcccgcggcc ccaaaaaccg aaataccagt gtgcagatct tggcccgcat ttacaagact
     9301 atcttgccag aaaaaaagcg tcgcagcagg tcatcaaaaa ttttaaatgg ctagagactt
     9361 atcgaaagca gcgagacagg cgcgaaggtg ccaccagatt cgcacgcggc ggccccagcg
     9421 cccaggccag gcctcaactc aagcacgagg cgaaggggct ccttaagcgc aaggcctcga
     9481 actctcccac ccacttccaa cccgaagctc gggatcaaga atcacgtact gcagccaggt
     9541 ggaagtaatt caaggcacgc aagggccata acccgtaaag aggccaggcc cgcgggaacc
     9601 acacacggca cttacctgtg ttctggcggc aaacccgttg cgaaaaagaa cgttcacggc
     9661 gactactgca cttatatacg gttctccccc accctcggga aaaaggcgga gccagtacac
     9721 gacatcactt tcccagttta ccccgcgcca ccttctctag gcaccggttc aattgccgac
     9781 ccctcccccc aacttctcgg ggactgtggg cgatgtgcgc tctgcccact gacgggcacc
     9841 ggagccaatt cccactcctt tcaagaccta gaaggtccat tagctgcaaa gattcctctc
     9901 tgtttaaaac tttatccatc tttgcagcta gctaattaac caaactggat ctctgctgtc
     9961 cctgtaataa acccgaaaat tttgaatttt tgtaatttgt ttttgtaatt ctttagtttg
    10021 tatgtctgtt gctattatgt ctactattct ttcccctgca ctgtaccccc caatcccccc
    10081 ttttctttta aaattgtgga tgaatactgc catttgtctg cagaattggc gcacgcagtg
    10141 ccgatccgtt cactaatcga atggatctgt ctctgtctct ctctccacct tcttcttcta
    10201 ttccttcggg cctgtcgggt cccctcgggg ttgggaggtg ggtctgaaac gataatggtg
    10261 aatatccctg cctaactcta ttcactatag aaagtacagc aaaaactatt cttaaaccta
    10321 ccaagcctcc tactatcatt atgaataatt ttatatacca cagccaattt gttatgttaa
    10381 accaattcca caaacttgcc catttatcta attccaataa ttcttgttca ttcttttctt
    10441 gctggttttg cgattcttca attaaggagt gtattaagct tgtgtaattg ttaatttctc
    10501 tgtcccactc catccaggtc gtgtgattcc aaatctgttc cagagattta ttactccaac
    10561 tagcattcca aggcacagca gtggtgcaaa tgagttttcc agagcaaccc caaatcccca
    10621 ggagctgttg atcctttagg tatctttcca cagccaggat tcttgcctgg agctgcttga
    10681 tgccccagac tgtgagttgc aacagatgct gttgcgcctc aatagccctc agcaaattgt
    10741 tctgctgctg cactatacca gacaataatt gtctggcctg taccgtcagc gtcattgacg
    10801 ctgcgcccat agtgcttcct gctgctccca agaacccaag gaacaaagct cctattccca
    10861 ctgctctttt ttctctctgc accactcttc tctttgcctt ggtgggtgct actcctaatg
    10921 gttcaatttt tactacttta tatttatata attcacttct ccaattgtcc ctcatatctc
    10981 ctcctccagg tctgaagatc agcggccgct tgctgtgcgg tggtcttact tttgttttgc
    11041 tcttcctcta tcttgtctaa agcttccttg gtgtctttta tctctatcct ttgatgcaca
    11101 caatagaggg ttgctactgt attatataat gatctaagtt cttctgatcc tgtctgaagg
    11161 gatggttgta gctgtcccag tatttgtcta cagccttctg atgtttctaa caggccagga
    11221 ttaactgcga atcgttctag ctccctgctt gcccatacta tatgttttaa tttatatttt
    11281 ttctttcccc ctggccttaa ccgaattttt tcccatcgcg atctaattct cccccgctta
    11341 atactgacgc tctcgcaccc atctctctcc ttctagcctc cgctagtcaa aatttttggc
    11401 gtactcacca gtcgccgccc ctcgcctctt gccgtgcgcg cttcagcaag ccgagtcctg
    11461 cgtcgagaga gctcctctgg tttccctttc gctttcaagt ccctgttcgg gcgccactgc
    11521 tagagatttt ccacactgac taaaagggtc tgagggatct ctagttacca gagtcacaca
    11581 acagacgggc acacactact tgaagcactc aaggcaagct ttattgaggc ttaagcagtg
    11641 ggttccctag ttagccagag agctcccagg ctcagatctg gtctaaccag agagacccag
    11701 tacaggcaaa acgcgctgct tatatagacc tcccaccgta cacgcctacc gcccatttgc
    11761 gtcaatgggg cggagttgtt acgacatttt ggaaagtccc gttgattttg gtgccaaaac
    11821 aaactcccat tgacgtcaat ggggtggaga cttggaaatc cccgtgagtc aaaccgctat
    11881 ccacgcccat tgatgtactg ccaaaaccgc atcaccatgg taatagcgat gactaatacg
    11941 tagatgtact gccaagtagg aaagtcccat aaggtcatgt actgggcata atgccaggcg
    12001 ggccatttac cgtcattgac gtcaataggg ggcgtacttg gcatatgata cacttgatgt
    12061 actgccaagt gggcagttta ccgtaaatac tccacccatt gacgtcaatg gaaagtccct
    12121 attggcgtta ctatgggaac atacgtcatt attgacgtca atgggcgggg gtcgttgggc
    12181 ggtcagccag gcgggccatt taccgtaagt tatgtaacgc ggaactccat atatgggcta
    12241 tgaactaatg accccgtaat tgattactat taataactag tcaataatca atgtcaacgc
    12301 gtatatctgg cccgtacatc gcgaagcagc gcaaaacgcc taaccctaag cagattcttc
    12361 atgcaattgt cggtcaagcc ttgccttgtt gtagcttaaa ttttgctcgc gcactactca
    12421 gcgacctcca acacacaagc agggagcaga tactggctta actatgcggc atcagagcag
    12481 attgtactga gagtgcacca taggggatcg ggagatctcc cgatccgtcg acgtcaggtg
    12541 gcacttttcg gggaaatgtg cgcggaaccc ctatttgttt atttttctaa atacattcaa
    12601 atatgtatcc gctcatgaga caataaccct gataaatgct tcaataatat tgaaaaagga
    12661 agagtatgag tattcaacat ttccgtgtcg cccttattcc cttttttgcg gcattttgcc
    12721 ttcctgtttt tgctcaccca gaaacgctgg tgaaagtaaa agatgctgaa gatcagttgg
    12781 gtgcacgagt gggttacatc gaactggatc tcaacagcgg taagatcctt gagagttttc
    12841 gccccgaaga acgttttcca atgatgagca cttttaaagt tctgctatgt ggcgcggtat
    12901 tatcccgtat tgacgccggg caagagcaac tcggtcgccg catacactat tctcagaatg
    12961 acttggttga gtactcacca gtcacagaaa agcatcttac ggatggcatg acagtaagag
    13021 aattatgcag tgctgccata accatgagtg ataacactgc ggccaactta cttctgacaa
    13081 cgatcggagg accgaaggag ctaaccgctt ttttgcacaa catgggggat catgtaactc
    13141 gccttgatcg ttgggaaccg gagctgaatg aagccatacc aaacgacgag cgtgacacca
    13201 cgatgcctgt agcaatggca acaacgttgc gcaaactatt aactggcgaa ctacttactc
    13261 tagcttcccg gcaacaatta atagactgga tggaggcgga taaagttgca ggaccacttc
    13321 tgcgctcggc ccttccggct ggctggttta ttgctgataa atctggagcc ggtgagcgtg
    13381 ggtctcgcgg tatcattgca gcactggggc cagatggtaa gccctcccgt atcgtagtta
    13441 tctacacgac ggggagtcag gcaactatgg atgaacgaaa tagacagatc gctgagatag
    13501 gtgcctcact gattaagcat tggtaactgt cagaccaagt ttactcatat atactttaga
    13561 ttgatttaaa acttcatttt taatttaaaa ggatctaggt gaagatcctt tttgataatc
    13621 tcatgaccaa aatcccttaa cgtgagtttt cgttccactg agcgtcagac cccgtagaaa
    13681 agatcaaagg atcttcttga gatccttttt ttctgcgcgt aatctgctgc ttgcaaacaa
    13741 aaaaaccacc gctaccagcg gtggtttgtt tgccggatca agagctacca actctttttc
    13801 cgaaggtaac tggcttcagc agagcgcaga taccaaatac tgttcttcta gtgtagccgt
    13861 agttaggcca ccacttcaag aactctgtag caccgcctac atacctcgct ctgctaatcc
    13921 tgttaccagt ggctgctgcc agtggcgata agtcgtgtct taccgggttg gactcaagac
    13981 gatagttacc ggataaggcg cagcggtcgg gctgaacggg gggttcgtgc acacagccca
    14041 gcttggagcg aacgacctac accgaactga gatacctaca gcgtgagcta tgagaaagcg
    14101 ccacgcttcc cgaagggaga aaggcggaca ggtatccggt aagcggcagg gtcggaacag
    14161 gagagcgcac gagggagctt ccagggggaa acgcctggta tctttatagt cctgtcgggt
    14221 ttcgccacct ctgacttgag cgtcgatttt tgtgatgctc gtcagggggg cggagcctat
    14281 ggaaaaacgc cagcaacgcg gcctttttac ggttcctggc cttttgctgg ccttttgctc
    14341 acatgttctt tcctgcgtta tcccctgatt ctgtggataa ccgtattacc gcctttgagt
    14401 gagctgatac cgctcgccgc agccgaacga ccgagcgcag cgagtcagtg agcgaggaag
    14461 cggaagagcg cccaatacgc aaaccgcctc tccccgcgcg ttggccgatt cattaatgca
    14521 gctggcacga caggtttccc gactggaaag cgggcagtga gcgcaacgca attaatgtga
    14581 gttagctcac tcattaggca ccccaggctt tacactttat gcttccggct cgtatgttgt
    14641 gtggaattgt gagcggataa caatttcaca caggaaacag ctatgaccat gattacgcca
    14701 agctctagct agaggtcgac ggtatacaga catgataaga tacattgatg agtttggaca
    14761 aaccacaact agaatgcagt gaaaaaaatg ctttatttgt gaaatttgtg atgctattgc
    14821 tttatttgta accattataa gctgcaataa acaagttggg gtgggcgaag aactccagca
    14881 tgagatcccc gcgctggagg atcatccagc cggcgtcccg gaaaacgatt ccgaagccca
    14941 acctttcata gaaggcggcg gtggaatcga aatctcgtag cacgtgtcag tcctgctcct
    15001 cggccacgaa gtgcacgcag ttgccggccg ggtcgcgcag ggcgaactcc cgcccccacg
    15061 gctgctcgcc gatctcggtc atggccggcc cggaggcgtc ccggaagttc gtggacacga
    15121 cctccgacca ctcggcgtac agctcgtcca ggccgcgcac ccacacccag gccagggtgt
    15181 tgtccggcac cacctggtcc tggaccgcgc tgatgaacag ggtcacgtcg tcccggacca
    15241 caccggcgaa gtcgtcctcc acgaagtccc gggagaaccc gagccggtcg gtccagaact
    15301 cgaccgctcc ggcgacgtcg cgcgcggtga gcaccggaac ggcactggtc aacttggcca
    15361 tggtttagtt cctcaccttg tcgtattata ctatgccgat atactatgcc gatgattaat
    15421 tgtcaacacg tgctgatcag atccgaaaat ggatatacaa gctcccggga gctttttgca
    15481 aaagcctagg cctccaaaaa agcctcctca cta
//