LOCUS       sequence-269065-       14651 bp    DNA     circular SYN 14-APR-2025
DEFINITION  Expresses human codon-optimized inactive SpCas9 fused to a 
            transcriptional activator VPR in mammalian cells. For cloning of 
            sgRNAs using BsmBI. Contains a barcode downstream of sgRNA 
            cassette..
ACCESSION   .
VERSION     .
KEYWORDS    .
SOURCE      synthetic DNA construct
  ORGANISM  synthetic DNA construct
REFERENCE   1  (bases 1 to 14651)
  AUTHORS   Legut M, Daniloski Z, Xue X, McKenzie D, Guo X, Wessels HH, Sanjana 
            NE
  TITLE     High-Throughput Screens of PAM-Flexible Cas9 Variants for Gene 
            Knockout and Transcriptional Modulation.
  JOURNAL   Cell Rep. 2020 Mar 3;30(9):2859-2868.e5. doi: 
            10.1016/j.celrep.2020.02.010.
  PUBMED    32130891
REFERENCE   2  (bases 1 to 14651)
  AUTHORS   .
  TITLE     Direct Submission
  JOURNAL   Exported Apr 14, 2025 from SnapGene Server 8.0.1
            https://www.snapgene.com
COMMENT     SGRef: number: 1; type: "Journal Article"; doi: 
            "10.1016/j.celrep.2020.02"; journalName: "Cell Rep"; date: 
            "2020-03-3- 3"; volume: "30"; issue: "9"; pages: "2859-2868.e5"
COMMENT     Sequence Label: pCC_05 -
            hU6-BsmBI-sgRNA(E+F)-barcode-EFS-dCas...
FEATURES             Location/Qualifiers
     source          1..14651
                     /mol_type="other DNA"
                     /organism="synthetic DNA construct"
     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"
     promoter        2608..2856
                     /label=U6 promoter
                     /note="RNA polymerase III promoter for human U6 snRNA 
                     (Domitrovich & Kunkel, 2003)"
     primer_bind     2608..2628
                     /label=hU6-F
                     /note="Human U6 promoter, forward primer"
     primer_bind     2779..2798
                     /label=LKO.1 5'
                     /note="Human U6 promoter, forward primer"
     promoter        3027..3238
                     /label=EF-1-alpha core promoter
                     /note="core promoter for human elongation factor 
                     EF-1-alpha"
     regulatory      3257..3266
                     /label=Kozak sequence
                     /note="vertebrate consensus sequence for strong initiation 
                     of translation (Kozak, 1987)"
                     /regulatory_class="other"
     CDS             join(3263..3289,3290..3292,3293..5779,5780..5782,
                     5783..7366)
                     /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             3263..7366
                     /codon_start=1
                     /product="Cas9 (Csn1) endonuclease from the Streptococcus 
                     pyogenes Type II CRISPR/Cas system"
                     /label=Cas9
                     /note="generates RNA-guided double strand breaks in DNA"
                     /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             7379..7399
                     /codon_start=1
                     /product="nuclear localization signal of SV40 (simian virus
                     40) large T antigen"
                     /label=SV40 NLS
                     /translation="PKKKRKV"
     protein_bind    7436..7460
                     /gene="mutant version of attB"
                     /label=attB1
                     /bound_moiety="BP Clonase(TM)"
                     /note="recombination site for the Gateway(R) BP reaction"
     CDS             7490..7639
                     /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             7664..7684
                     /codon_start=1
                     /product="nuclear localization signal of SV40 (simian virus
                     40) large T antigen"
                     /label=SV40 NLS
                     /translation="PKKKRKV"
     CDS             8114..8470
                     /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             8489..9058
                     /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             9074..9130
                     /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             9131..9727
                     /codon_start=1
                     /gene="pac from Streptomyces alboniger"
                     /product="puromycin N-acetyltransferase"
                     /label=PuroR
                     /note="confers resistance to puromycin"
                     /translation="TEYKPTVRLATRDDVPRAVRTLAAAFADYPATRHTVDPDRHIERV
                     TELQELFLTRVGLDIGKVWVADDGAAVAVWTTPESVEAGAVFAEIGPRMAELSGSRLAA
                     QQQMEGLLAPHRPKEPAWFLATVGVSPDHQGKGLGSAVVLPGVEAAERAGVPAFLETSA
                     PRNLPFYERLGFTVTADVEVPEGPRTWCMTRKPGA"
     primer_bind     9624..9644
                     /label=Puro-F
                     /note="Puromycin resistance gene, forward primer"
     misc_feature    9743..10331
                     /label=WPRE
                     /note="woodchuck hepatitis virus posttranscriptional 
                     regulatory element"
     primer_bind     complement(9796..9816)
                     /label=WPRE-R
                     /note="WPRE, reverse primer"
     CDS             complement(10214..10225)
                     /codon_start=1
                     /product="Factor Xa recognition and cleavage site"
                     /label=Factor Xa site
                     /translation="IEGR"
     LTR             10403..10636
                     /label=3' LTR (Delta-U3)
                     /note="self-inactivating 3' long terminal repeat (LTR) from
                     HIV-1"
     primer_bind     complement(10662..10679)
                     /label=BGH-rev
                     /note="Bovine growth hormone terminator, reverse primer. 
                     Also called BGH reverse"
     polyA_signal    10668..10892
                     /label=bGH poly(A) signal
                     /note="bovine growth hormone polyadenylation signal"
     rep_origin      10938..11366
                     /direction=RIGHT
                     /label=f1 ori
                     /note="f1 bacteriophage origin of replication; arrow 
                     indicates direction of (+) strand synthesis"
     primer_bind     complement(11025..11044)
                     /label=F1ori-R
                     /note="F1 origin, reverse primer"
     primer_bind     11235..11256
                     /label=F1ori-F
                     /note="F1 origin, forward primer"
     primer_bind     complement(11375..11395)
                     /label=pBABE 3'
                     /note="SV40 enhancer, reverse primer for pBABE vectors"
     promoter        11380..11709
                     /label=SV40 promoter
                     /note="SV40 enhancer and early promoter"
     rep_origin      11560..11695
                     /label=SV40 ori
                     /note="SV40 origin of replication"
     primer_bind     11622..11641
                     /label=SV40pro-F
                     /note="SV40 promoter/origin, forward primer"
     promoter        11757..11804
                     /label=EM7 promoter
                     /note="synthetic bacterial promoter "
     CDS             11823..12197
                     /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    12327..12448
                     /label=SV40 poly(A) signal
                     /note="SV40 polyadenylation signal"
     primer_bind     complement(12364..12383)
                     /label=SV40pA-R
                     /note="SV40 polyA, reverse primer"
     primer_bind     12418..12437
                     /label=EBV-rev
                     /note="SV40 polyA terminator, reverse primer"
     primer_bind     complement(12497..12513)
                     /label=M13 rev
                     /note="common sequencing primer, one of multiple similar 
                     variants"
     primer_bind     complement(12497..12513)
                     /label=M13 Reverse
                     /note="In lacZ gene. Also called M13-rev"
     primer_bind     complement(12510..12532)
                     /label=M13/pUC Reverse
                     /note="In lacZ gene"
     protein_bind    12521..12537
                     /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(join(12545..12551,12552..12569,12570..12575))
                     /label=lac promoter
                     /note="promoter for the E. coli lac operon"
     protein_bind    12590..12611
                     /label=CAP binding site
                     /bound_moiety="E. coli catabolite activator protein"
                     /note="CAP binding activates transcription in the presence 
                     of cAMP."
     primer_bind     complement(12728..12745)
                     /label=L4440
                     /note="L4440 vector, forward primer"
     rep_origin      complement(12899..13487)
                     /direction=LEFT
                     /label=ori
                     /note="high-copy-number ColE1/pMB1/pBR322/pUC origin of 
                     replication"
     primer_bind     complement(12979..12998)
                     /label=pBR322ori-F
                     /note="pBR322 origin, forward primer"
     CDS             complement(join(13658..14449,14450..14518))
                     /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     14281..14300
                     /label=Amp-R
                     /note="Ampicillin resistance gene, reverse primer"
     promoter        complement(14519..14623)
                     /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 aggtaccgag ggcctatttc ccatgattcc ttcatatttg
     2641 catatacgat acaaggctgt tagagagata attagaatta atttgactgt aaacacaaag
     2701 atattagtac aaaatacgtg acgtagaaag taataatttc ttgggtagtt tgcagtttta
     2761 aaattatgtt ttaaaatgga ctatcatatg cttaccgtaa cttgaaagta tttcgatttc
     2821 ttggctttat atatcttgtg gaaaggacga aacaccggag acggacgtct ctgtttaaga
     2881 gctatgctgg aaacagcata gcaagtttaa ataaggctag tccgttatca acttgaaaaa
     2941 gtggcaccga gtcggtgctt tttttgaatt cgggtcggct agctaggtct tgaaaggagt
     3001 gggaattggc tccggtgccc gtcagtgggc agagcgcaca tcgcccacag tccccgagaa
     3061 gttgggggga ggggtcggca attgatccgg tgcctagaga aggtggcgcg gggtaaactg
     3121 ggaaagtgat gtcgtgtact ggctccgcct ttttcccgag ggtgggggag aaccgtatat
     3181 aagtgcagta gtcgccgtga acgttctttt tcgcaacggg tttgccgcca gaacacagga
     3241 ccggttctag agcgctgcca ccatggacaa gaagtacagc atcggcctgg ccatcggcac
     3301 caactctgtg ggctgggccg tgatcaccga cgagtacaag gtgcccagca agaaattcaa
     3361 ggtgctgggc aacaccgacc ggcacagcat caagaagaac ctgatcggcg ccctgctgtt
     3421 cgacagcgga gaaacagccg aggccacccg gctgaagaga accgccagaa gaagatacac
     3481 cagacggaag aaccggatct gctatctgca agagatcttc agcaacgaga tggccaaggt
     3541 ggacgacagc ttcttccaca gactggaaga gtccttcctg gtggaagagg ataagaagca
     3601 cgagcggcac cccatcttcg gcaacatcgt ggacgaggtg gcctaccacg agaagtaccc
     3661 caccatctac cacctgagaa agaaactggt ggacagcacc gacaaggccg acctgcggct
     3721 gatctatctg gccctggccc acatgatcaa gttccggggc cacttcctga tcgagggcga
     3781 cctgaacccc gacaacagcg acgtggacaa gctgttcatc cagctggtgc agacctacaa
     3841 ccagctgttc gaggaaaacc ccatcaacgc cagcggcgtg gacgccaagg ccatcctgtc
     3901 tgccagactg agcaagagca gacggctgga aaatctgatc gcccagctgc ccggcgagaa
     3961 gaagaatggc ctgttcggca acctgattgc cctgagcctg ggcctgaccc ccaacttcaa
     4021 gagcaacttc gacctggccg aggatgccaa actgcagctg agcaaggaca cctacgacga
     4081 cgacctggac aacctgctgg cccagatcgg cgaccagtac gccgacctgt ttctggccgc
     4141 caagaacctg tccgacgcca tcctgctgag cgacatcctg agagtgaaca ccgagatcac
     4201 caaggccccc ctgagcgcct ctatgatcaa gagatacgac gagcaccacc aggacctgac
     4261 cctgctgaaa gctctcgtgc ggcagcagct gcctgagaag tacaaagaga ttttcttcga
     4321 ccagagcaag aacggctacg ccggctacat cgatggcgga gccagccagg aagagttcta
     4381 caagttcatc aagcccatcc tggaaaagat ggacggcacc gaggaactgc tcgtgaagct
     4441 gaacagagag gacctgctgc ggaagcagcg gaccttcgac aacggcagca tcccccacca
     4501 gatccacctg ggagagctgc acgccattct gcggcggcag gaagattttt acccattcct
     4561 gaaggacaac cgggaaaaga tcgagaagat cctgaccttc cgcatcccct actacgtggg
     4621 ccctctggcc aggggaaaca gcagattcgc ctggatgacc agaaagagcg aggaaaccat
     4681 caccccctgg aacttcgagg aagtggtgga caagggcgcc agcgcccaga gcttcatcga
     4741 gcggatgacc aacttcgata agaacctgcc caacgagaag gtgctgccca agcacagcct
     4801 gctgtacgag tacttcaccg tgtacaacga gctgaccaaa gtgaaatacg tgaccgaggg
     4861 aatgagaaag cccgccttcc tgagcggcga gcagaaaaaa gccatcgtgg acctgctgtt
     4921 caagaccaac cggaaagtga ccgtgaagca gctgaaagag gactacttca agaaaatcga
     4981 gtgcttcgac tccgtggaaa tctccggcgt ggaagatcgg ttcaacgcct ccctgggcac
     5041 ataccacgat ctgctgaaaa ttatcaagga caaggacttc ctggacaatg aggaaaacga
     5101 ggacattctg gaagatatcg tgctgaccct gacactgttt gaggacagag agatgatcga
     5161 ggaacggctg aaaacctatg cccacctgtt cgacgacaaa gtgatgaagc agctgaagcg
     5221 gcggagatac accggctggg gcaggctgag ccggaagctg atcaacggca tccgggacaa
     5281 gcagtccggc aagacaatcc tggatttcct gaagtccgac ggcttcgcca acagaaactt
     5341 catgcagctg atccacgacg acagcctgac ctttaaagag gacatccaga aagcccaggt
     5401 gtccggccag ggcgatagcc tgcacgagca cattgccaat ctggccggca gccccgccat
     5461 taagaagggc atcctgcaga cagtgaaggt ggtggacgag ctcgtgaaag tgatgggccg
     5521 gcacaagccc gagaacatcg tgatcgaaat ggccagagag aaccagacca cccagaaggg
     5581 acagaagaac agccgcgaga gaatgaagcg gatcgaagag ggcatcaaag agctgggcag
     5641 ccagatcctg aaagaacacc ccgtggaaaa cacccagctg cagaacgaga agctgtacct
     5701 gtactacctg cagaatgggc gggatatgta cgtggaccag gaactggaca tcaaccggct
     5761 gtccgactac gatgtggacg ctatcgtgcc tcagagcttt ctgaaggacg actccatcga
     5821 taacaaagtg ctgactcgga gcgacaagaa ccggggcaag agcgacaacg tgccctccga
     5881 agaggtcgtg aagaagatga agaactactg gcgccagctg ctgaatgcca agctgattac
     5941 ccagaggaag ttcgacaatc tgaccaaggc cgagagaggc ggcctgagcg aactggataa
     6001 ggccggcttc atcaagagac agctggtgga aacccggcag atcacaaagc acgtggcaca
     6061 gatcctggac tcccggatga acactaagta cgacgagaac gacaaactga tccgggaagt
     6121 gaaagtgatc accctgaagt ccaagctggt gtccgatttc cggaaggatt tccagtttta
     6181 caaagtgcgc gagatcaaca actaccacca cgcccacgac gcctacctga acgccgtcgt
     6241 gggaaccgcc ctgatcaaaa agtaccctaa gctggaaagc gagttcgtgt acggcgacta
     6301 caaggtgtac gacgtgcgga agatgatcgc caagagcgag caggaaatcg gcaaggctac
     6361 cgccaagtac ttcttctaca gcaacatcat gaactttttc aagaccgaga ttaccctggc
     6421 caacggcgag atccggaagc ggcctctgat cgagacaaac ggcgaaacag gcgagatcgt
     6481 gtgggataag ggccgggact ttgccaccgt gcggaaagtg ctgtctatgc cccaagtgaa
     6541 tatcgtgaaa aagaccgagg tgcagacagg cggcttcagc aaagagtcta tcctgcccaa
     6601 gaggaacagc gacaagctga tcgccagaaa gaaggactgg gaccctaaga agtacggcgg
     6661 cttcgacagc cccaccgtgg cctattctgt gctggtggtg gccaaagtgg aaaagggcaa
     6721 gtccaagaaa ctgaagagtg tgaaagagct gctggggatc accatcatgg aaagaagcag
     6781 cttcgagaag aatcccatcg actttctgga agccaagggc tacaaagaag tgaaaaagga
     6841 cctgatcatc aagctgccta agtactccct gttcgagctg gaaaacggcc ggaagagaat
     6901 gctggcctct gccggcgaac tgcagaaggg aaacgaactg gccctgccct ccaaatatgt
     6961 gaacttcctg tacctggcca gccactatga gaagctgaag ggctcccccg aggataatga
     7021 gcagaaacag ctgtttgtgg aacagcacaa acactacctg gacgagatca tcgagcagat
     7081 cagcgagttc tccaagagag tgatcctggc cgacgctaat ctggacaagg tgctgagcgc
     7141 ctacaacaag cacagagaca agcctatcag agagcaggcc gagaatatca tccacctgtt
     7201 taccctgacc aatctgggag cccctgccgc cttcaagtac tttgacacca ccatcgaccg
     7261 gaagaggtac accagcacca aagaggtgct ggacgccacc ctgatccacc agagcatcac
     7321 cggcctgtac gagacacgga tcgacctgtc tcagctggga ggcgacagca gggctgaccc
     7381 caagaagaag aggaaggtgt cgccagggat ccgtcgactt gacgcgttga tatcaacaag
     7441 tttgtacaaa aaagcaggct acaaagaggc cagcggttcc ggacgggctg acgcattgga
     7501 cgattttgat ctggatatgc tgggaagtga cgccctcgat gattttgacc ttgacatgct
     7561 tggttcggat gcccttgatg actttgacct cgacatgctc ggcagtgacg cccttgatga
     7621 tttcgacctg gacatgctga ttaactctag aagttccgga tctccgaaaa agaaacgcaa
     7681 agttggtagc cagtacctgc ccgacaccga cgaccggcac cggatcgagg aaaagcggaa
     7741 gcggacctac gagacattca agagcatcat gaagaagtcc cccttcagcg gccccaccga
     7801 ccctagacct ccacctagaa gaatcgccgt gcccagcaga tccagcgcca gcgtgccaaa
     7861 acctgccccc cagccttacc ccttcaccag cagcctgagc accatcaact acgacgagtt
     7921 ccctaccatg gtgttcccca gcggccagat ctctcaggcc tctgctctgg ctccagcccc
     7981 tcctcaggtg ctgcctcagg ctcctgctcc tgcaccagct ccagccatgg tgtctgcact
     8041 ggctcaggca ccagcacccg tgcctgtgct ggctcctgga cctccacagg ctgtggctcc
     8101 accagcccct aaacctacac aggccggcga gggcacactg tctgaagctc tgctgcagct
     8161 gcagttcgac gacgaggatc tgggagccct gctgggaaac agcaccgatc ctgccgtgtt
     8221 caccgacctg gccagcgtgg acaacagcga gttccagcag ctgctgaacc agggcatccc
     8281 tgtggcccct cacaccaccg agcccatgct gatggaatac cccgaggcca tcacccggct
     8341 cgtgacaggc gctcagaggc ctcctgatcc agctcctgcc cctctgggag caccaggcct
     8401 gcctaatgga ctgctgtctg gcgacgagga cttcagctct atcgccgata tggatttctc
     8461 agccttgctg ggctctggca gcggcagccg ggattccagg gaagggatgt ttttgccgaa
     8521 gcctgaggcc ggctccgcta ttagtgacgt gtttgagggc cgcgaggtgt gccagccaaa
     8581 acgaatccgg ccatttcatc ctccaggaag tccatgggcc aaccgcccac tccccgccag
     8641 cctcgcacca acaccaaccg gtccagtaca tgagccagtc gggtcactga ccccggcacc
     8701 agtccctcag ccactggatc cagcgcccgc agtgactccc gaggccagtc acctgttgga
     8761 ggatcccgat gaagaaacga gccaggctgt caaagccctt cgggagatgg ccgatactgt
     8821 gattccccag aaggaagagg ctgcaatctg tggccaaatg gacctttccc atccgccccc
     8881 aaggggccat ctggatgagc tgacaaccac acttgagtcc atgaccgagg atctgaacct
     8941 ggactcaccc ctgaccccgg aattgaacga gattctggat accttcctga acgacgagtg
     9001 cctcttgcat gccatgcata tcagcacagg actgtccatc ttcgacacat ctctctttga
     9061 attcggatcc ggcgcaacaa acttctctct gctgaaacaa gccggagatg tcgaagagaa
     9121 tcctggaccg accgagtaca agcccacggt gcgcctcgcc acccgcgacg acgtccccag
     9181 ggccgtacgc accctcgccg ccgcgttcgc cgactacccc gccacgcgcc acaccgtcga
     9241 tccggaccgc cacatcgagc gggtcaccga gctgcaagaa ctcttcctca cgcgcgtcgg
     9301 gctcgacatc ggcaaggtgt gggtcgcgga cgacggcgcc gcggtggcgg tctggaccac
     9361 gccggagagc gtcgaagcgg gggcggtgtt cgccgagatc ggcccgcgca tggccgagtt
     9421 gagcggttcc cggctggccg cgcagcaaca gatggaaggc ctcctggcgc cgcaccggcc
     9481 caaggagccc gcgtggttcc tggccaccgt cggagtctcg cccgaccacc agggcaaggg
     9541 tctgggcagc gccgtcgtgc tccccggagt ggaggcggcc gagcgcgccg gggtgcccgc
     9601 cttcctggag acctccgcgc cccgcaacct ccccttctac gagcggctcg gcttcaccgt
     9661 caccgccgac gtcgaggtgc ccgaaggacc gcgcacctgg tgcatgaccc gcaagcccgg
     9721 tgcctgaacg cgttaagtcg acaatcaacc tctggattac aaaatttgtg aaagattgac
     9781 tggtattctt aactatgttg ctccttttac gctatgtgga tacgctgctt taatgccttt
     9841 gtatcatgct attgcttccc gtatggcttt cattttctcc tccttgtata aatcctggtt
     9901 gctgtctctt tatgaggagt tgtggcccgt tgtcaggcaa cgtggcgtgg tgtgcactgt
     9961 gtttgctgac gcaaccccca ctggttgggg cattgccacc acctgtcagc tcctttccgg
    10021 gactttcgct ttccccctcc ctattgccac ggcggaactc atcgccgcct gccttgcccg
    10081 ctgctggaca ggggctcggc tgttgggcac tgacaattcc gtggtgttgt cggggaaatc
    10141 atcgtccttt ccttggctgc tcgcctgtgt tgccacctgg attctgcgcg ggacgtcctt
    10201 ctgctacgtc ccttcggccc tcaatccagc ggaccttcct tcccgcggcc tgctgccggc
    10261 tctgcggcct cttccgcgtc ttcgccttcg ccctcagacg agtcggatct ccctttgggc
    10321 cgcctccccg cgtcgacttt aagaccaatg acttacaagg cagctgtaga tcttagccac
    10381 tttttaaaag aaaagggggg actggaaggg ctaattcact cccaacgaag acaagatctg
    10441 ctttttgctt gtactgggtc tctctggtta gaccagatct gagcctggga gctctctggc
    10501 taactaggga acccactgct taagcctcaa taaagcttgc cttgagtgct tcaagtagtg
    10561 tgtgcccgtc tgttgtgtga ctctggtaac tagagatccc tcagaccctt ttagtcagtg
    10621 tggaaaatct ctagcagggc ccgtttaaac ccgctgatca gcctcgactg tgccttctag
    10681 ttgccagcca tctgttgttt gcccctcccc cgtgccttcc ttgaccctgg aaggtgccac
    10741 tcccactgtc ctttcctaat aaaatgagga aattgcatcg cattgtctga gtaggtgtca
    10801 ttctattctg gggggtgggg tggggcagga cagcaagggg gaggattggg aagacaatag
    10861 caggcatgct ggggatgcgg tgggctctat ggcttctgag gcggaaagaa ccagctgggg
    10921 ctctaggggg tatccccacg cgccctgtag cggcgcatta agcgcggcgg gtgtggtggt
    10981 tacgcgcagc gtgaccgcta cacttgccag cgccctagcg cccgctcctt tcgctttctt
    11041 cccttccttt ctcgccacgt tcgccggctt tccccgtcaa gctctaaatc gggggctccc
    11101 tttagggttc cgatttagtg ctttacggca cctcgacccc aaaaaacttg attagggtga
    11161 tggttcacgt agtgggccat cgccctgata gacggttttt cgccctttga cgttggagtc
    11221 cacgttcttt aatagtggac tcttgttcca aactggaaca acactcaacc ctatctcggt
    11281 ctattctttt gatttataag ggattttgcc gatttcggcc tattggttaa aaaatgagct
    11341 gatttaacaa aaatttaacg cgaattaatt ctgtggaatg tgtgtcagtt agggtgtgga
    11401 aagtccccag gctccccagc aggcagaagt atgcaaagca tgcatctcaa ttagtcagca
    11461 accaggtgtg gaaagtcccc aggctcccca gcaggcagaa gtatgcaaag catgcatctc
    11521 aattagtcag caaccatagt cccgccccta actccgccca tcccgcccct aactccgccc
    11581 agttccgccc attctccgcc ccatggctga ctaatttttt ttatttatgc agaggccgag
    11641 gccgcctctg cctctgagct attccagaag tagtgaggag gcttttttgg aggcctaggc
    11701 ttttgcaaaa agctcccggg agcttgtata tccattttcg gatctgatca gcacgtgttg
    11761 acaattaatc atcggcatag tatatcggca tagtataata cgacaaggtg aggaactaaa
    11821 ccatggccaa gttgaccagt gccgttccgg tgctcaccgc gcgcgacgtc gccggagcgg
    11881 tcgagttctg gaccgaccgg ctcgggttct cccgggactt cgtggaggac gacttcgccg
    11941 gtgtggtccg ggacgacgtg accctgttca tcagcgcggt ccaggaccag gtggtgccgg
    12001 acaacaccct ggcctgggtg tgggtgcgcg gcctggacga gctgtacgcc gagtggtcgg
    12061 aggtcgtgtc cacgaacttc cgggacgcct ccgggccggc catgaccgag atcggcgagc
    12121 agccgtgggg gcgggagttc gccctgcgcg acccggccgg caactgcgtg cacttcgtgg
    12181 ccgaggagca ggactgacac gtgctacgag atttcgattc caccgccgcc ttctatgaaa
    12241 ggttgggctt cggaatcgtt ttccgggacg ccggctggat gatcctccag cgcggggatc
    12301 tcatgctgga gttcttcgcc caccccaact tgtttattgc agcttataat ggttacaaat
    12361 aaagcaatag catcacaaat ttcacaaata aagcattttt ttcactgcat tctagttgtg
    12421 gtttgtccaa actcatcaat gtatcttatc atgtctgtat accgtcgacc tctagctaga
    12481 gcttggcgta atcatggtca tagctgtttc ctgtgtgaaa ttgttatccg ctcacaattc
    12541 cacacaacat acgagccgga agcataaagt gtaaagcctg gggtgcctaa tgagtgagct
    12601 aactcacatt aattgcgttg cgctcactgc ccgctttcca gtcgggaaac ctgtcgtgcc
    12661 agctgcatta atgaatcggc caacgcgcgg ggagaggcgg tttgcgtatt gggcgctctt
    12721 ccgcttcctc gctcactgac tcgctgcgct cggtcgttcg gctgcggcga gcggtatcag
    12781 ctcactcaaa ggcggtaata cggttatcca cagaatcagg ggataacgca ggaaagaaca
    12841 tgtgagcaaa aggccagcaa aaggccagga accgtaaaaa ggccgcgttg ctggcgtttt
    12901 tccataggct ccgcccccct gacgagcatc acaaaaatcg acgctcaagt cagaggtggc
    12961 gaaacccgac aggactataa agataccagg cgtttccccc tggaagctcc ctcgtgcgct
    13021 ctcctgttcc gaccctgccg cttaccggat acctgtccgc ctttctccct tcgggaagcg
    13081 tggcgctttc tcatagctca cgctgtaggt atctcagttc ggtgtaggtc gttcgctcca
    13141 agctgggctg tgtgcacgaa ccccccgttc agcccgaccg ctgcgcctta tccggtaact
    13201 atcgtcttga gtccaacccg gtaagacacg acttatcgcc actggcagca gccactggta
    13261 acaggattag cagagcgagg tatgtaggcg gtgctacaga gttcttgaag tggtggccta
    13321 actacggcta cactagaaga acagtatttg gtatctgcgc tctgctgaag ccagttacct
    13381 tcggaaaaag agttggtagc tcttgatccg gcaaacaaac caccgctggt agcggtggtt
    13441 tttttgtttg caagcagcag attacgcgca gaaaaaaagg atctcaagaa gatcctttga
    13501 tcttttctac ggggtctgac gctcagtgga acgaaaactc acgttaaggg attttggtca
    13561 tgagattatc aaaaaggatc ttcacctaga tccttttaaa ttaaaaatga agttttaaat
    13621 caatctaaag tatatatgag taaacttggt ctgacagtta ccaatgctta atcagtgagg
    13681 cacctatctc agcgatctgt ctatttcgtt catccatagt tgcctgactc cccgtcgtgt
    13741 agataactac gatacgggag ggcttaccat ctggccccag tgctgcaatg ataccgcgag
    13801 acccacgctc accggctcca gatttatcag caataaacca gccagccgga agggccgagc
    13861 gcagaagtgg tcctgcaact ttatccgcct ccatccagtc tattaattgt tgccgggaag
    13921 ctagagtaag tagttcgcca gttaatagtt tgcgcaacgt tgttgccatt gctacaggca
    13981 tcgtggtgtc acgctcgtcg tttggtatgg cttcattcag ctccggttcc caacgatcaa
    14041 ggcgagttac atgatccccc atgttgtgca aaaaagcggt tagctccttc ggtcctccga
    14101 tcgttgtcag aagtaagttg gccgcagtgt tatcactcat ggttatggca gcactgcata
    14161 attctcttac tgtcatgcca tccgtaagat gcttttctgt gactggtgag tactcaacca
    14221 agtcattctg agaatagtgt atgcggcgac cgagttgctc ttgcccggcg tcaatacggg
    14281 ataataccgc gccacatagc agaactttaa aagtgctcat cattggaaaa cgttcttcgg
    14341 ggcgaaaact ctcaaggatc ttaccgctgt tgagatccag ttcgatgtaa cccactcgtg
    14401 cacccaactg atcttcagca tcttttactt tcaccagcgt ttctgggtga gcaaaaacag
    14461 gaaggcaaaa tgccgcaaaa aagggaataa gggcgacacg gaaatgttga atactcatac
    14521 tcttcctttt tcaatattat tgaagcattt atcagggtta ttgtctcatg agcggataca
    14581 tatttgaatg tatttagaaa aataaacaaa taggggttcc gcgcacattt ccccgaaaag
    14641 tgccacctga c
//
