dCas9-VPR_P2A_mCherry

LOCUS        dCas9-VPR_P2A_mCherry      14796 bp    DNA     circular SYN 14-APR-2025
DEFINITION  Lentiviral expression plasmid encoding dCas9-VPR and a P2A linked 
            mCherry marker.
ACCESSION   .
VERSION     .
KEYWORDS    .
SOURCE      synthetic DNA construct
  ORGANISM  synthetic DNA construct
REFERENCE   1  (bases 1 to 14796)
  AUTHORS   Umkehrer C, Holstein F, Formenti L, Jude J, Froussios K, Neumann T, 
            Cronin SM, Haas L, Lipp JJ, Burkard TR, Fellner M, Wiesner T, Zuber 
            J, Obenauf AC
  TITLE     Isolating live cell clones from barcoded populations using 
            CRISPRa-inducible reporters.
  JOURNAL   Nat Biotechnol. 2020 Jul 27. pii: 10.1038/s41587-020-0614-0. doi: 
            10.1038/s41587-020-0614-0.
  PUBMED    32719478
REFERENCE   2  (bases 1 to 14796)
  AUTHORS   .
  TITLE     Direct Submission
  JOURNAL   Exported Apr 14, 2025 from SnapGene Server 8.0.1
            https://www.biofeng.com
COMMENT     SGRef: number: 1; type: "Journal Article"; journalName: "Nat 
            Biotechnol. 2020 Jul 27. pii: 10.1038/s41587-020-0614-0. doi: 
            10.1038/s41587-020-0614-0."
COMMENT     Sequence Label: dCas9-VPR_P2A_mCherry
FEATURES             Location/Qualifiers
     source          1..14796
                     /mol_type="other DNA"
                     /organism="synthetic DNA construct"
     promoter        complement(335..546)
                     /label=EF-1-alpha core promoter
                     /note="core promoter for human elongation factor 
                     EF-1-alpha"
     misc_feature    639..756
                     /label=cPPT/CTS
                     /note="central polypurine tract and central termination 
                     sequence of HIV-1"
     CDS             complement(1054..1098)
                     /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    1283..1516
                     /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    2009..2134
                     /label=HIV-1 Psi
                     /note="packaging signal of human immunodeficiency virus 
                     type 1"
     LTR             2181..2361
                     /label=5' LTR (truncated)
                     /note="truncated 5' long terminal repeat (LTR) from HIV-1"
     promoter        complement(2379..2577)
                     /label=CMV promoter
                     /note="human cytomegalovirus (CMV) immediate early 
                     promoter"
     primer_bind     complement(2404..2424)
                     /label=CMV-F
                     /note="Human CMV immediate early promoter, forward primer"
     enhancer        2579..2958
                     /label=CMV enhancer
                     /note="human cytomegalovirus immediate early enhancer"
     primer_bind     3130..3149
                     /label=pRS-marker
                     /note="pRS vectors, use to sequence yeast selectable 
                     marker"
     promoter        3224..3328
                     /gene="bla"
                     /label=AmpR promoter
     CDS             join(3329..3397,3398..4189)
                     /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(3547..3566)
                     /label=Amp-R
                     /note="Ampicillin resistance gene, reverse primer"
     rep_origin      4360..4948
                     /direction=RIGHT
                     /label=ori
                     /note="high-copy-number ColE1/pMB1/pBR322/pUC origin of 
                     replication"
     primer_bind     4849..4868
                     /label=pBR322ori-F
                     /note="pBR322 origin, forward primer"
     primer_bind     5102..5119
                     /label=L4440
                     /note="L4440 vector, forward primer"
     protein_bind    5236..5257
                     /label=CAP binding site
                     /bound_moiety="E. coli catabolite activator protein"
                     /note="CAP binding activates transcription in the presence 
                     of cAMP."
     promoter        join(5272..5277,5278..5295,5296..5302)
                     /label=lac promoter
                     /note="promoter for the E. coli lac operon"
     protein_bind    5310..5326
                     /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     5315..5337
                     /label=M13/pUC Reverse
                     /note="In lacZ gene"
     primer_bind     5334..5350
                     /label=M13 rev
                     /note="common sequencing primer, one of multiple similar 
                     variants"
     primer_bind     5334..5350
                     /label=M13 Reverse
                     /note="In lacZ gene. Also called M13-rev"
     polyA_signal    5399..5520
                     /label=SV40 poly(A) signal
                     /note="SV40 polyadenylation signal"
     primer_bind     complement(5410..5429)
                     /label=EBV-rev
                     /note="SV40 polyA terminator, reverse primer"
     primer_bind     5464..5483
                     /label=SV40pA-R
                     /note="SV40 polyA, reverse primer"
     CDS             complement(5650..6024)
                     /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(6043..6090)
                     /label=EM7 promoter
                     /note="synthetic bacterial promoter "
     promoter        complement(6138..6467)
                     /label=SV40 promoter
                     /note="SV40 enhancer and early promoter"
     rep_origin      6152..6287
                     /label=SV40 ori
                     /note="SV40 origin of replication"
     primer_bind     complement(6206..6225)
                     /label=SV40pro-F
                     /note="SV40 promoter/origin, forward primer"
     primer_bind     6452..6472
                     /label=pBABE 3'
                     /note="SV40 enhancer, reverse primer for pBABE vectors"
     rep_origin      complement(6481..6909)
                     /direction=LEFT
                     /label=f1 ori
                     /note="f1 bacteriophage origin of replication; arrow 
                     indicates direction of (+) strand synthesis"
     primer_bind     complement(6591..6612)
                     /label=F1ori-F
                     /note="F1 origin, forward primer"
     primer_bind     6803..6822
                     /label=F1ori-R
                     /note="F1 origin, reverse primer"
     polyA_signal    6955..7179
                     /label=bGH poly(A) signal
                     /note="bovine growth hormone polyadenylation signal"
     primer_bind     7168..7185
                     /label=BGH-rev
                     /note="Bovine growth hormone terminator, reverse primer. 
                     Also called BGH reverse"
     LTR             7211..7391
                     /label=5' LTR (truncated)
                     /note="truncated 5' long terminal repeat (LTR) from HIV-1"
     primer_bind     7896..7912
                     /label=pBluescriptKS
                     /note="For pBluescript vector"
     primer_bind     7897..7913
                     /label=KS primer
                     /note="common sequencing primer, one of multiple similar 
                     variants"
     misc_feature    7916..8504
                     /label=WPRE
                     /note="woodchuck hepatitis virus posttranscriptional 
                     regulatory element"
     CDS             8022..8033
                     /codon_start=1
                     /product="Factor Xa recognition and cleavage site"
                     /label=Factor Xa site
                     /translation="IEGR"
     primer_bind     8431..8451
                     /label=WPRE-R
                     /note="WPRE, reverse primer"
     CDS             complement(8529..9239)
                     /codon_start=1
                     /product="monomeric derivative of DsRed fluorescent protein
                     (Shaner et al., 2004)"
                     /label=mCherry
                     /note="mammalian codon-optimized"
                     /translation="MVSKGEEDNMAIIKEFMRFKVHMEGSVNGHEFEIEGEGEGRPYEG
                     TQTAKLKVTKGGPLPFAWDILSPQFMYGSKAYVKHPADIPDYLKLSFPEGFKWERVMNF
                     EDGGVVTVTQDSSLQDGEFIYKVKLRGTNFPSDGPVMQKKTMGWEASSERMYPEDGALK
                     GEIKQRLKLKDGGHYDAEVKTTYKAKKPVQLPGAYNVNIKLDITSHNEDYTIVEQYERA
                     EGRHSTGGMDELYK"
     primer_bind     complement(8807..8826)
                     /label=mCherry-F
                     /note="mCherry, forward primer"
     primer_bind     9076..9094
                     /label=mCherry-R
                     /note="mCherry, reverse primer"
     CDS             complement(9240..9296)
                     /codon_start=1
                     /product="2A peptide from porcine teschovirus-1 
                     polyprotein"
                     /label=P2A
                     /note="Eukaryotic ribosomes fail to insert a peptide bond 
                     between the Gly and Pro residues, yielding separate 
                     polypeptides."
                     /translation="ATNFSLLKQAGDVEENPGP"
     CDS             complement(9312..9881)
                     /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(9900..10256)
                     /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(10686..10706)
                     /codon_start=1
                     /product="nuclear localization signal of SV40 (simian virus
                     40) large T antigen"
                     /label=SV40 NLS
                     /translation="PKKKRKV"
     CDS             complement(10731..10880)
                     /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"
     protein_bind    complement(10910..10934)
                     /gene="mutant version of attB"
                     /label=attB1
                     /bound_moiety="BP Clonase(TM)"
                     /note="recombination site for the Gateway(R) BP reaction"
     CDS             complement(10971..10991)
                     /codon_start=1
                     /product="nuclear localization signal of SV40 (simian virus
                     40) large T antigen"
                     /label=SV40 NLS
                     /translation="PKKKRKV"
     CDS             complement(join(11004..12518,12519..12521,12522..12587,
                     12588..12593,12594..14796,1..281,282..284,285..311))
                     /codon_start=1
                     /product="catalytically dead mutant of the Cas9 
                     endonuclease from the Streptococcus pyogenes Type II 
                     CRISPR/Cas system"
                     /label=Cas9m4
                     /note="RNA-guided DNA-binding protein that lacks 
                     endonuclease activity due to the mutations D10A, D839A, 
                     H840A, and N863A (Mali et al., 2013)"
                     /translation="MDKKYSIGLAIGTNSVGWAVITDEYKVPSKKFKVLGNTDRHSIKK
                     NLIGALLFDSGETAEATRLKRTARRRYTRRKNRICYLQEIFSNEMAKVDDSFFHRLEES
                     FLVEEDKKHERHPIFGNIVDEVAYHEKYPTIYHLRKKLVDSTDKADLRLIYLALAHMIK
                     FRGHFLIEGDLNPDNSDVDKLFIQLVQTYNQLFEENPINASGVDAKAILSARLSKSRRL
                     ENLIAQLPGEKKNGLFGNLIALSLGLTPNFKSNFDLAEDAKLQLSKDTYDDDLDNLLAQ
                     IGDQYADLFLAAKNLSDAILLSDILRVNTEITKAPLSASMIKRYDEHHQDLTLLKALVR
                     QQLPEKYKEIFFDQSKNGYAGYIDGGASQEEFYKFIKPILEKMDGTEELLVKLNREDLL
                     RKQRTFDNGSIPHQIHLGELHAILRRQEDFYPFLKDNREKIEKILTFRIPYYVGPLARG
                     NSRFAWMTRKSEETITPWNFEEVVDKGASAQSFIERMTNFDKNLPNEKVLPKHSLLYEY
                     FTVYNELTKVKYVTEGMRKPAFLSGEQKKAIVDLLFKTNRKVTVKQLKEDYFKKIECFD
                     SVEISGVEDRFNASLGTYHDLLKIIKDKDFLDNEENEDILEDIVLTLTLFEDREMIEER
                     LKTYAHLFDDKVMKQLKRRRYTGWGRLSRKLINGIRDKQSGKTILDFLKSDGFANRNFM
                     QLIHDDSLTFKEDIQKAQVSGQGDSLHEHIANLAGSPAIKKGILQTVKVVDELVKVMGR
                     HKPENIVIEMARENQTTQKGQKNSRERMKRIEEGIKELGSQILKEHPVENTQLQNEKLY
                     LYYLQNGRDMYVDQELDINRLSDYDVAAIVPQSFLKDDSIDNKVLTRSDKARGKSDNVP
                     SEEVVKKMKNYWRQLLNAKLITQRKFDNLTKAERGGLSELDKAGFIKRQLVETRQITKH
                     VAQILDSRMNTKYDENDKLIREVKVITLKSKLVSDFRKDFQFYKVREINNYHHAHDAYL
                     NAVVGTALIKKYPKLESEFVYGDYKVYDVRKMIAKSEQEIGKATAKYFFYSNIMNFFKT
                     EITLANGEIRKRPLIETNGETGEIVWDKGRDFATVRKVLSMPQVNIVKKTEVQTGGFSK
                     ESILPKRNSDKLIARKKDWDPKKYGGFDSPTVAYSVLVVAKVEKGKSKKLKSVKELLGI
                     TIMERSSFEKNPIDFLEAKGYKEVKKDLIIKLPKYSLFELENGRKRMLASAGELQKGNE
                     LALPSKYVNFLYLASHYEKLKGSPEDNEQKQLFVEQHKHYLDEIIEQISEFSKRVILAD
                     ANLDKVLSAYNKHRDKPIREQAENIIHLFTLTNLGAPAAFKYFDTTIDRKRYTSTKEVL
                     DATLIHQSITGLYETRIDLSQLGGD"
ORIGIN
        1 gactcctcca gcctatggaa gaaagagtca tccaccttag ccatctcatt actaaagatc
       61 tcctgcaggt agcagatccg attctttctg cgggtatatc tgcgccgtgc tgttcttttg
      121 agccgcgtgg cttcggccgt ctccccggag tcgaacagga gggcgccaat gaggttcttc
      181 tttatgctgt ggcgatcggt attgcccaga actttgaatt ttttgctcgg caccttgtac
      241 tcgtccgtaa tgacggccca gccgacgctg tttgtgccga tagcgagccc aatggagtac
      301 ttcttgtcca tggtggagcg ctctagaacc ggtcctgtgt tctggcggca aacccgttgc
      361 gaaaaagaac gttcacggcg actactgcac ttatatacgg ttctccccca ccctcgggaa
      421 aaaggcggag ccagtacacg acatcacttt cccagtttac cccgcgccac cttctctagg
      481 caccggatca attgccgacc cctcccccca acttctcggg gactgtgggc gatgtgcgct
      541 ctgcccactg acgggcaccg gagccaattc ccactccttt caagacctag ctagctaatt
      601 aaccaaactg gatctctgct gtccctgtaa taaacccgaa aattttgaat ttttgtaatt
      661 tgtttttgta attctttagt ttgtatgtct gttgctatta tgtctactat tctttcccct
      721 gcactgtacc ccccaatccc cccttttctt ttaaaattgt ggatgaatac tgccatttgt
      781 ctgcagaatt ggcgcacgca gtgccgatcc gttcactaat cgaatggatc tgtctctgtc
      841 tctctctcca ccttcttctt ctattccttc gggcctgtcg ggtcccctcg gggttgggag
      901 gtgggtctga aacgataatg gtgaatatcc ctgcctaact ctattcacta tagaaagtac
      961 agcaaaaact attcttaaac ctaccaagcc tcctactatc attatgaata attttatata
     1021 ccacagccaa tttgttatgt taaaccaatt ccacaaactt gcccatttat ctaattccaa
     1081 taattcttgt tcattctttt cttgctggtt ttgcgattct tcaattaagg agtgtattaa
     1141 gcttgtgtaa ttgttaattt ctctgtccca ctccatccag gtcgtgtgat tccaaatctg
     1201 ttccagagat ttattactcc aactagcatt ccaaggcaca gcagtggtgc aaatgagttt
     1261 tccagagcaa ccccaaatcc ccaggagctg ttgatccttt aggtatcttt ccacagccag
     1321 gattcttgcc tggagctgct tgatgcccca gactgtgagt tgcaacagat gctgttgcgc
     1381 ctcaatagcc ctcagcaaat tgttctgctg ctgcactata ccagacaata attgtctggc
     1441 ctgtaccgtc agcgtcattg acgctgcgcc catagtgctt cctgctgctc ccaagaaccc
     1501 aaggaacaaa gctcctattc ccactgctct tttttctctc tgcaccactc ttctctttgc
     1561 cttggtgggt gctactccta atggttcaat ttttactact ttatatttat ataattcact
     1621 tctccaattg tccctcatat ctcctcctcc aggtctgaag atcagcggcc gcttgctgtg
     1681 cggtggtctt acttttgttt tgctcttcct ctatcttgtc taaagcttcc ttggtgtctt
     1741 ttatctctat cctttgatgc acacaataga gggttgctac tgtattatat aatgatctaa
     1801 gttcttctga tcctgtctga agggatggtt gtagctgtcc cagtatttgt ctacagcctt
     1861 ctgatgtttc taacaggcca ggattaactg cgaatcgttc tagctccctg cttgcccata
     1921 ctatatgttt taatttatat tttttctttc cccctggcct taaccgaatt ttttcccatc
     1981 gcgatctaat tctcccccgc ttaatactga cgctctcgca cccatctctc tccttctagc
     2041 ctccgctagt caaaattttt ggcgtactca ccagtcgccg cccctcgcct cttgccgtgc
     2101 gcgcttcagc aagccgagtc ctgcgtcgag agagctcctc tggtttccct ttcgctttca
     2161 agtccctgtt cgggcgccac tgctagagat tttccacact gactaaaagg gtctgaggga
     2221 tctctagtta ccagagtcac acaacagacg ggcacacact acttgaagca ctcaaggcaa
     2281 gctttattga ggcttaagca gtgggttccc tagttagcca gagagctccc aggctcagat
     2341 ctggtctaac cagagagacc cagtacaggc aaaacgcgct gcttatatag acctcccacc
     2401 gtacacgcct accgcccatt tgcgtcaatg gggcggagtt gttacgacat tttggaaagt
     2461 cccgttgatt ttggtgccaa aacaaactcc cattgacgtc aatggggtgg agacttggaa
     2521 atccccgtga gtcaaaccgc tatccacgcc cattgatgta ctgccaaaac cgcatcacca
     2581 tggtaatagc gatgactaat acgtagatgt actgccaagt aggaaagtcc cataaggtca
     2641 tgtactgggc ataatgccag gcgggccatt taccgtcatt gacgtcaata gggggcgtac
     2701 ttggcatatg atacacttga tgtactgcca agtgggcagt ttaccgtaaa tactccaccc
     2761 attgacgtca atggaaagtc cctattggcg ttactatggg aacatacgtc attattgacg
     2821 tcaatgggcg ggggtcgttg ggcggtcagc caggcgggcc atttaccgta agttatgtaa
     2881 cgcggaactc catatatggg ctatgaacta atgaccccgt aattgattac tattaataac
     2941 tagtcaataa tcaatgtcaa cgcgtatatc tggcccgtac atcgcgaagc agcgcaaaac
     3001 gcctaaccct aagcagattc ttcatgcaat tgtcggtcaa gccttgcctt gttgtagctt
     3061 aaattttgct cgcgcactac tcagcgacct ccaacacaca agcagggagc agatactggc
     3121 ttaactatgc ggcatcagag cagattgtac tgagagtgca ccatagggga tcgggagatc
     3181 tcccgatccg tcgacgtcag gtggcacttt tcggggaaat gtgcgcggaa cccctatttg
     3241 tttatttttc taaatacatt caaatatgta tccgctcatg agacaataac cctgataaat
     3301 gcttcaataa tattgaaaaa ggaagagtat gagtattcaa catttccgtg tcgcccttat
     3361 tccctttttt gcggcatttt gccttcctgt ttttgctcac ccagaaacgc tggtgaaagt
     3421 aaaagatgct gaagatcagt tgggtgcacg agtgggttac atcgaactgg atctcaacag
     3481 cggtaagatc cttgagagtt ttcgccccga agaacgtttt ccaatgatga gcacttttaa
     3541 agttctgcta tgtggcgcgg tattatcccg tattgacgcc gggcaagagc aactcggtcg
     3601 ccgcatacac tattctcaga atgacttggt tgagtactca ccagtcacag aaaagcatct
     3661 tacggatggc atgacagtaa gagaattatg cagtgctgcc ataaccatga gtgataacac
     3721 tgcggccaac ttacttctga caacgatcgg aggaccgaag gagctaaccg cttttttgca
     3781 caacatgggg gatcatgtaa ctcgccttga tcgttgggaa ccggagctga atgaagccat
     3841 accaaacgac gagcgtgaca ccacgatgcc tgtagcaatg gcaacaacgt tgcgcaaact
     3901 attaactggc gaactactta ctctagcttc ccggcaacaa ttaatagact ggatggaggc
     3961 ggataaagtt gcaggaccac ttctgcgctc ggcccttccg gctggctggt ttattgctga
     4021 taaatctgga gccggtgagc gtgggtctcg cggtatcatt gcagcactgg ggccagatgg
     4081 taagccctcc cgtatcgtag ttatctacac gacggggagt caggcaacta tggatgaacg
     4141 aaatagacag atcgctgaga taggtgcctc actgattaag cattggtaac tgtcagacca
     4201 agtttactca tatatacttt agattgattt aaaacttcat ttttaattta aaaggatcta
     4261 ggtgaagatc ctttttgata atctcatgac caaaatccct taacgtgagt tttcgttcca
     4321 ctgagcgtca gaccccgtag aaaagatcaa aggatcttct tgagatcctt tttttctgcg
     4381 cgtaatctgc tgcttgcaaa caaaaaaacc accgctacca gcggtggttt gtttgccgga
     4441 tcaagagcta ccaactcttt ttccgaaggt aactggcttc agcagagcgc agataccaaa
     4501 tactgttctt ctagtgtagc cgtagttagg ccaccacttc aagaactctg tagcaccgcc
     4561 tacatacctc gctctgctaa tcctgttacc agtggctgct gccagtggcg ataagtcgtg
     4621 tcttaccggg ttggactcaa gacgatagtt accggataag gcgcagcggt cgggctgaac
     4681 ggggggttcg tgcacacagc ccagcttgga gcgaacgacc tacaccgaac tgagatacct
     4741 acagcgtgag ctatgagaaa gcgccacgct tcccgaaggg agaaaggcgg acaggtatcc
     4801 ggtaagcggc agggtcggaa caggagagcg cacgagggag cttccagggg gaaacgcctg
     4861 gtatctttat agtcctgtcg ggtttcgcca cctctgactt gagcgtcgat ttttgtgatg
     4921 ctcgtcaggg gggcggagcc tatggaaaaa cgccagcaac gcggcctttt tacggttcct
     4981 ggccttttgc tggccttttg ctcacatgtt ctttcctgcg ttatcccctg attctgtgga
     5041 taaccgtatt accgcctttg agtgagctga taccgctcgc cgcagccgaa cgaccgagcg
     5101 cagcgagtca gtgagcgagg aagcggaaga gcgcccaata cgcaaaccgc ctctccccgc
     5161 gcgttggccg attcattaat gcagctggca cgacaggttt cccgactgga aagcgggcag
     5221 tgagcgcaac gcaattaatg tgagttagct cactcattag gcaccccagg ctttacactt
     5281 tatgcttccg gctcgtatgt tgtgtggaat tgtgagcgga taacaatttc acacaggaaa
     5341 cagctatgac catgattacg ccaagctcta gctagaggtc gacggtatac agacatgata
     5401 agatacattg atgagtttgg acaaaccaca actagaatgc agtgaaaaaa atgctttatt
     5461 tgtgaaattt gtgatgctat tgctttattt gtaaccatta taagctgcaa taaacaagtt
     5521 ggggtgggcg aagaactcca gcatgagatc cccgcgctgg aggatcatcc agccggcgtc
     5581 ccggaaaacg attccgaagc ccaacctttc atagaaggcg gcggtggaat cgaaatctcg
     5641 tagcacgtgt cagtcctgct cctcggccac gaagtgcacg cagttgccgg ccgggtcgcg
     5701 cagggcgaac tcccgccccc acggctgctc gccgatctcg gtcatggccg gcccggaggc
     5761 gtcccggaag ttcgtggaca cgacctccga ccactcggcg tacagctcgt ccaggccgcg
     5821 cacccacacc caggccaggg tgttgtccgg caccacctgg tcctggaccg cgctgatgaa
     5881 cagggtcacg tcgtcccgga ccacaccggc gaagtcgtcc tccacgaagt cccgggagaa
     5941 cccgagccgg tcggtccaga actcgaccgc tccggcgacg tcgcgcgcgg tgagcaccgg
     6001 aacggcactg gtcaacttgg ccatggttta gttcctcacc ttgtcgtatt atactatgcc
     6061 gatatactat gccgatgatt aattgtcaac acgtgctgat cagatccgaa aatggatata
     6121 caagctcccg ggagcttttt gcaaaagcct aggcctccaa aaaagcctcc tcactacttc
     6181 tggaatagct cagaggcaga ggcggcctcg gcctctgcat aaataaaaaa aattagtcag
     6241 ccatggggcg gagaatgggc ggaactgggc ggagttaggg gcgggatggg cggagttagg
     6301 ggcgggacta tggttgctga ctaattgaga tgcatgcttt gcatacttct gcctgctggg
     6361 gagcctgggg actttccaca cctggttgct gactaattga gatgcatgct ttgcatactt
     6421 ctgcctgctg gggagcctgg ggactttcca caccctaact gacacacatt ccacagaatt
     6481 aattcgcgtt aaatttttgt taaatcagct cattttttaa ccaataggcc gaaatcggca
     6541 aaatccctta taaatcaaaa gaatagaccg agatagggtt gagtgttgtt ccagtttgga
     6601 acaagagtcc actattaaag aacgtggact ccaacgtcaa agggcgaaaa accgtctatc
     6661 agggcgatgg cccactacgt gaaccatcac cctaatcaag ttttttgggg tcgaggtgcc
     6721 gtaaagcact aaatcggaac cctaaaggga gcccccgatt tagagcttga cggggaaagc
     6781 cggcgaacgt ggcgagaaag gaagggaaga aagcgaaagg agcgggcgct agggcgctgg
     6841 caagtgtagc ggtcacgctg cgcgtaacca ccacacccgc cgcgcttaat gcgccgctac
     6901 agggcgcgtg gggatacccc ctagagcccc agctggttct ttccgcctca gaagccatag
     6961 agcccaccgc atccccagca tgcctgctat tgtcttccca atcctccccc ttgctgtcct
     7021 gccccacccc accccccaga atagaatgac acctactcag acaatgcgat gcaatttcct
     7081 cattttatta ggaaaggaca gtgggagtgg caccttccag ggtcaaggaa ggcacggggg
     7141 aggggcaaac aacagatggc tggcaactag aaggcacagt cgaggctgat cagcgggttt
     7201 aaacgggccc tgctagagat tttccacact gactaaaagg gtctgaggga tctctagtta
     7261 ccagagtcac acaacagacg ggcacacact acttgaagca ctcaaggcaa gctttattga
     7321 ggcttaagca gtgggttccc tagttagcca gagagctccc aggctcagat ctggtctaac
     7381 cagagagacc cagtacagtc cggatgcagc tctcgggcca tgtgatgaaa tgctaggcgg
     7441 ctgtcaaacc tccactctaa tacttctctc tccgggtcat ccatcccatg caggctcaca
     7501 gggtgtaaca agcgggtgtt ctctccttca ttggcttctt ctaccttctc ttgctcaact
     7561 ggtactagct tgtagcacca tccaaaggtc agtggatatc tgatccctgg ccctggtgtg
     7621 tagttctgcc aatcagggaa gtagccttgt gtgtggtaga tccacagatc aaggatatct
     7681 tgtcttcgtt gggagtgaat tagcccttcc agtcccccct tttcttttaa aaagtggcta
     7741 agatctacag ctgccttgta agtcattggt cttaaaggta cctgaggtgt gactggaaaa
     7801 cccacctcct cctcctcttg tgcttctagc caggcacaat cagcattggt agctgctgta
     7861 ttgctacttg tgattgctcc atgtttttct aggtctcgag gtcgacggta tcgatgcggg
     7921 gaggcggccc aaagggagat ccgactcgtc tgagggcgaa ggcgaagacg cggaagaggc
     7981 cgcagagccg gcagcaggcc gcgggaagga aggtccgctg gattgagggc cgaagggacg
     8041 tagcagaagg acgtcccgcg cagaatccag gtggcaacac aggcgagcag ccaaggaaag
     8101 gacgatgatt tccccgacaa caccacggaa ttgtcagtgc ccaacagccg agcccctgtc
     8161 cagcagcggg caaggcaggc ggcgatgagt tccgccgtgg caatagggag ggggaaagcg
     8221 aaagtcccgg aaaggagctg acaggtggtg gcaatgcccc aaccagtggg ggttgcgtca
     8281 gcaaacacag tgcacaccac gccacgttgc ctgacaacgg gccacaactc ctcataaaga
     8341 gacagcaacc aggatttata caaggaggag aaaatgaaag ccatacggga agcaatagca
     8401 tgatacaaag gcattaaagc agcgtatcca catagcgtaa aaggagcaac atagttaaga
     8461 ataccagtca atctttcaca aattttgtaa tccagaggtt gattaccgat aagcttgata
     8521 tcgaattctc acttgtacag ctcgtccatg ccgccggtgg agtggcggcc ctcggcgcgt
     8581 tcgtactgtt ccacgatggt gtagtcctcg ttgtgggagg tgatgtccaa cttgatgttg
     8641 acgttgtagg cgccgggcag ctgcacgggc ttcttggcct tgtaggtggt cttgacctca
     8701 gcgtcgtagt ggccgccgtc cttcagcttc agcctctgct tgatctcgcc cttcagggcg
     8761 ccgtcctcgg ggtacatccg ctcggaggag gcctcccagc ccatggtctt cttctgcatt
     8821 acggggccgt cggaggggaa gttggtgccg cgcagcttca ccttgtagat gaactcgccg
     8881 tcctgcaggg aggagtcctg ggtcacggtc accacgccgc cgtcctcgaa gttcatcacg
     8941 cgctcccatt tgaagccctc ggggaaggac agcttcaagt agtcggggat gtcggcgggg
     9001 tgcttcacgt aggccttgga gccgtacatg aactgagggg acaggatgtc ccaggcgaag
     9061 ggcagggggc cacccttggt caccttcagc ttggcggtct gggtgccctc gtaggggcgg
     9121 ccctcgccct cgccctcgat ctcgaactcg tggccgttca cggagccctc catgtgcacc
     9181 ttgaagcgca tgaactcctt gatgatggcc atgttatcct cctcgccctt gctcaccatg
     9241 gggccggggt tctcctccac gtcgccggcc tgcttcagca ggctgaagtt ggtggcgccg
     9301 ctgccggatc caaacagaga tgtgtcgaag atggacagtc ctgtgctgat atgcatggca
     9361 tgcaagaggc actcgtcgtt caggaaggta tccagaatct cgttcaattc cggggtcagg
     9421 ggtgagtcca ggttcagatc ctcggtcatg gactcaagtg tggttgtcag ctcatccaga
     9481 tggccccttg ggggcggatg ggaaaggtcc atttggccac agattgcagc ctcttccttc
     9541 tggggaatca cagtatcggc catctcccga agggctttga cagcctggct cgtctcttca
     9601 tcgggatcct ccaacaggtg actggcctcg ggagtcactg cgggcgctgg atccagtggc
     9661 tgagggactg gtgccggggt cagtgacccg actggctcat gtactggacc ggttggtgtt
     9721 ggtgcgaggc tggcggggag tgggcggttg gcccatggac ttcctggagg atgaaatggc
     9781 cggattcgtt ttggctggca cacctcgcgg ccctcaaaca cgtcactaat agcggagccg
     9841 gcctcaggct tcggcaaaaa catcccttcc ctggaatccc ggctgccgct gccagagccc
     9901 agcaaggctg agaaatccat atcggcgata gagctgaagt cctcgtcgcc agacagcagt
     9961 ccattaggca ggcctggtgc tcccagaggg gcaggagctg gatcaggagg cctctgagcg
    10021 cctgtcacga gccgggtgat ggcctcgggg tattccatca gcatgggctc ggtggtgtga
    10081 ggggccacag ggatgccctg gttcagcagc tgctggaact cgctgttgtc cacgctggcc
    10141 aggtcggtga acacggcagg atcggtgctg tttcccagca gggctcccag atcctcgtcg
    10201 tcgaactgca gctgcagcag agcttcagac agtgtgccct cgccggcctg tgtaggttta
    10261 ggggctggtg gagccacagc ctgtggaggt ccaggagcca gcacaggcac gggtgctggt
    10321 gcctgagcca gtgcagacac catggctgga gctggtgcag gagcaggagc ctgaggcagc
    10381 acctgaggag gggctggagc cagagcagag gcctgagaga tctggccgct ggggaacacc
    10441 atggtaggga actcgtcgta gttgatggtg ctcaggctgc tggtgaaggg gtaaggctgg
    10501 ggggcaggtt ttggcacgct ggcgctggat ctgctgggca cggcgattct tctaggtgga
    10561 ggtctagggt cggtggggcc gctgaagggg gacttcttca tgatgctctt gaatgtctcg
    10621 taggtccgct tccgcttttc ctcgatccgg tgccggtcgt cggtgtcggg caggtactgg
    10681 ctaccaactt tgcgtttctt tttcggagat ccggaacttc tagagttaat cagcatgtcc
    10741 aggtcgaaat catcaagggc gtcactgccg agcatgtcga ggtcaaagtc atcaagggca
    10801 tccgaaccaa gcatgtcaag gtcaaaatca tcgagggcgt cacttcccag catatccaga
    10861 tcaaaatcgt ccaatgcgtc agcccgtccg gaaccgctgg cctctttgta gcctgctttt
    10921 ttgtacaaac ttgttgatat caacgcgtca agtcgacgga tccctggcga caccttcctc
    10981 ttcttcttgg ggtcagccct gctgtctcca ccgagctgag agaggtcgat tcttgtttca
    11041 tagagccccg taattgactg atgaatcagt gtggcgtcca ggacctcctt tgtagaggtg
    11101 taccgctttc tgtctatggt ggtgtcgaag tacttgaagg ctgcaggcgc gcccaagttg
    11161 gtcagagtaa acaagtggat aatgttttct gcctgctccc tgatgggctt atccctgtgc
    11221 ttattgtaag cagaaagcac cttatcgagg ttagcgtcgg cgaggatcac tcttttggag
    11281 aattcgctta tttgctcgat gatctcatca aggtagtgtt tgtgttgttc cacgaacagc
    11341 tgcttctgct cattatcttc gggagaccct ttgagctttt catagtggct ggccagatac
    11401 aagaaattaa cgtatttaga gggcagtgcc agctcgttac ctttctgcag ctcgcccgca
    11461 ctagcgagca ttcgtttccg gccgttttca agctcaaaga gagagtactt gggaagctta
    11521 atgatgaggt cttttttgac ctctttatat cctttcgcct cgagaaagtc gatggggttt
    11581 ttttcgaagc ttgatcgctc catgattgtg atgcccagca gttccttgac gcttttgagt
    11641 tttttagact tccctttctc cactttggcc acaaccagta cactgtaagc gactgtagga
    11701 gaatcgaatc cgccgtattt cttggggtcc caatcttttt tgcgtgcgat cagcttgtcg
    11761 ctgttccttt tcgggaggat actttccttg gagaagcctc cggtctgtac ttcggtcttt
    11821 ttaacgatgt tcacctgcgg catggacagg accttccgga ctgtcgcgaa atccctaccc
    11881 ttgtcccaca cgatttctcc tgtttctccg tttgtttcga taagtggtcg cttccgaatc
    11941 tctccattgg ccagtgtaat ctcggtcttg aaaaaattca taatattgct gtaaaagaag
    12001 tacttagcgg tggccttgcc tatttcctgc tcagactttg cgatcatttt cctaacatcg
    12061 tacactttat agtctccgta aacaaattca gattcaagct tgggatattt tttgataagt
    12121 gcagtgccta ccactgcatt caggtaggca tcatgcgcat ggtggtaatt gttgatctct
    12181 ctcaccttat aaaactgaaa gtcctttctg aaatctgaga ccagcttaga cttcagagta
    12241 ataactttca cctctcgaat cagtttgtca ttttcatcgt acttggtgtt catgcgtgaa
    12301 tcgagaattt gggccacgtg cttggtgatc tggcgtgtct caacaagctg ccttttgatg
    12361 aagccggctt tatccaactc agacaggcca cctcgttcag ccttagtcag attatcgaac
    12421 ttccgttgtg tgatcagttt ggcgttcagc agctgccgcc aataattttt cattttcttg
    12481 acaacttctt ctgaggggac gttatcactc ttccctctag ctttatcgga tcttgtcaac
    12541 actttattat caatagaatc atctttgaga aaagactggg gcacgatagc agccacgtcg
    12601 tagtcggaga gccgattgat gtccagttcc tgatccacgt acatgtccct gccgttctgc
    12661 aggtagtaca ggtagagctt ctcattctga agctgggtgt tttcaactgg gtgttcctta
    12721 aggatttggg accccagttc ttttataccc tcttcaatcc tcttcatcct ttccctactg
    12781 ttcttctgtc ccttctgggt agtttggttc tctcgggcca tctcgataac gatattctcg
    12841 ggcttatgcc ttcccattac tttgacgagt tcatccacga ccttaacggt ctgcagtatt
    12901 ccctttttga tagctgggct acctgcaaga ttagcgatgt gctcgtgaag actgtccccc
    12961 tggccagaaa cttgtgcttt ctggatgtcc tccttaaagg tgagagagtc atcatggatc
    13021 aactgcatga agttccggtt ggcaaatcca tcggacttaa gaaaatccag gattgtcttt
    13081 ccactctgct tgtctcggat cccattgatc agttttcttg acagccgccc ccatcctgta
    13141 tatcggcgcc tcttgagctg tttcatgact ttgtcgtcga agagatgagc gtaagttttc
    13201 aagcgttctt caatcatctc cctatcttca aacaacgtaa gggtgaggac aatgtcctca
    13261 agaatgtcct cgttctcctc attgtccagg aagtccttgt ctttaatgat tttcaggaga
    13321 tcgtgatacg ttcccaggga tgcgttgaag cgatcctcca ctccgctgat ttcaacagag
    13381 tcgaaacatt caatcttttt gaaatagtct tctttgagct gtttcacggt aactttccgg
    13441 ttcgtcttga agaggaggtc cacgatagct ttcttctgct ctccagacag gaatgctggc
    13501 tttctcatcc cttctgtgac gtatttgacc ttggtgagct cgttataaac tgtgaagtac
    13561 tcgtacagca gagagtgttt aggaagcacc ttttcgttag gcagattttt atcaaagtta
    13621 gtcatccttt cgatgaagga ctgggcagag gcccccttat ccacgacttc ctcgaagttc
    13681 cagggagtga tggtctcttc tgatttgcga gtcatccacg cgaatctgga atttccccgg
    13741 gcgagggggc ctacatagta gggtatccga aatgtgagga ttttctcaat cttttccctg
    13801 ttatctttca aaaaggggta gaaatcctct tgccgcctga ggatagcgtg cagttcgccc
    13861 aggtgaatct ggtgggggat gcttccattg tcgaaagtgc gctgtttgcg caacagatct
    13921 tctctgttaa gctttaccag cagctcctcg gtgccgtcca ttttttccaa gatgggctta
    13981 ataaatttgt aaaattcctc ctggcttgct ccgccgtcaa tgtatccggc gtagccattt
    14041 ttagactgat cgaagaaaat ttccttgtac ttctcaggca gttgctgtct gacaagggcc
    14101 ttcagcaaag tcaagtcttg gtggtgctca tcatagcgct tgatcatact agcgctcagc
    14161 ggagctttgg tgatctccgt gttcactcgc agaatatcac tcagcagaat ggcgtctgac
    14221 aggttctttg ccgccaaaaa aaggtctgcg tactggtcgc cgatctgggc cagcagattg
    14281 tcgagatcat catcgtaggt gtctttgctc agttgaagct tggcatcttc ggccaggtcg
    14341 aagttagatt taaagttggg ggtcagcccg agtgacaggg cgataagatt accaaacagg
    14401 ccgttcttct tctccccagg gagctgtgcg atgaggtttt cgagccgccg ggatttggac
    14461 agcctagcgc tcaggattgc tttggcgtca actccggatg cgttgatcgg gttctcttcg
    14521 aaaagctgat tgtaagtctg aaccagttgg ataaagagtt tgtcgacatc gctgttgtct
    14581 gggttcaggt ccccctcgat gaggaagtgt ccccgaaatt tgatcatatg cgccagcgcg
    14641 agatagatca accgcaagtc agccttatca gtactgtcta caagcttctt cctcagatga
    14701 tatatggttg ggtacttttc atggtacgcc acctcgtcca cgatattgcc aaagattggg
    14761 tggcgctcgt gctttttatc ctcctccacc aaaaag
//