pFUGW SpCas9

LOCUS       pFUGW SpCas9               13418 bp ds-DNA     circular SYN 12-MAY-2021
DEFINITION  lentiviral expression of SpCas9 under hUBC promotor.
ACCESSION   .
VERSION     .
KEYWORDS    pFUGW SpCas9
SOURCE      synthetic DNA construct
  ORGANISM  synthetic DNA construct
REFERENCE   1  (bases 1 to 13418)
  AUTHORS   Willems J, de Jong APH, Scheefhals N, Mertens E, Catsburg LAE, 
            Poorthuis RB, de Winter F, Verhaagen J, Meye FJ, MacGillavry HD
  TITLE     ORANGE: A CRISPR/Cas9-based genome editing toolbox for epitope 
            tagging of endogenous proteins in neurons.
  JOURNAL   PLoS Biol. 2020 Apr 10;18(4):e3000665. doi: 
            10.1371/journal.pbio.3000665.
  PUBMED    32275651
REFERENCE   2  (bases 1 to 13418)
  AUTHORS   .
  TITLE     Direct Submission
  JOURNAL   Exported May 12, 2021 from SnapGene Server 1.1.58
            http://www.biofeng.com
FEATURES             Location/Qualifiers
     source          1..13418
                     /organism="synthetic DNA construct"
                     /mol_type="other DNA"
     primer_bind     262..281
                     /label=hUBCpro-F
                     /note="Human Ubiquitin C (UbC) promoter, forward primer"
     CDS             481..507
                     /codon_start=1
                     /product="HA (human influenza hemagglutinin) epitope tag"
                     /label=HA
                     /translation="YPYDVPDYA"
     CDS             511..531
                     /codon_start=1
                     /product="nuclear localization signal of SV40 (simian virus
                     40) large T antigen"
                     /label=SV40 NLS
                     /translation="PKKKRKV"
     CDS             541..4641
                     /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="DKKYSIGLDIGTNSVGWAVITDEYKVPSKKFKVLGNTDRHSIKKN
                     LIGALLFDSGETAEATRLKRTARRRYTRRKNRICYLQEIFSNEMAKVDDSFFHRLEESF
                     LVEEDKKHERHPIFGNIVDEVAYHEKYPTIYHLRKKLVDSTDKADLRLIYLALAHMIKF
                     RGHFLIEGDLNPDNSDVDKLFIQLVQTYNQLFEENPINASGVDAKAILSARLSKSRRLE
                     NLIAQLPGEKKNGLFGNLIALSLGLTPNFKSNFDLAEDAKLQLSKDTYDDDLDNLLAQI
                     GDQYADLFLAAKNLSDAILLSDILRVNTEITKAPLSASMIKRYDEHHQDLTLLKALVRQ
                     QLPEKYKEIFFDQSKNGYAGYIDGGASQEEFYKFIKPILEKMDGTEELLVKLNREDLLR
                     KQRTFDNGSIPHQIHLGELHAILRRQEDFYPFLKDNREKIEKILTFRIPYYVGPLARGN
                     SRFAWMTRKSEETITPWNFEEVVDKGASAQSFIERMTNFDKNLPNEKVLPKHSLLYEYF
                     TVYNELTKVKYVTEGMRKPAFLSGEQKKAIVDLLFKTNRKVTVKQLKEDYFKKIECFDS
                     VEISGVEDRFNASLGTYHDLLKIIKDKDFLDNEENEDILEDIVLTLTLFEDREMIEERL
                     KTYAHLFDDKVMKQLKRRRYTGWGRLSRKLINGIRDKQSGKTILDFLKSDGFANRNFMQ
                     LIHDDSLTFKEDIQKAQVSGQGDSLHEHIANLAGSPAIKKGILQTVKVVDELVKVMGRH
                     KPENIVIEMARENQTTQKGQKNSRERMKRIEEGIKELGSQILKEHPVENTQLQNEKLYL
                     YYLQNGRDMYVDQELDINRLSDYDVDHIVPQSFLKDDSIDNKVLTRSDKNRGKSDNVPS
                     EEVVKKMKNYWRQLLNAKLITQRKFDNLTKAERGGLSELDKAGFIKRQLVETRQITKHV
                     AQILDSRMNTKYDENDKLIREVKVITLKSKLVSDFRKDFQFYKVREINNYHHAHDAYLN
                     AVVGTALIKKYPKLESEFVYGDYKVYDVRKMIAKSEQEIGKATAKYFFYSNIMNFFKTE
                     ITLANGEIRKRPLIETNGETGEIVWDKGRDFATVRKVLSMPQVNIVKKTEVQTGGFSKE
                     SILPKRNSDKLIARKKDWDPKKYGGFDSPTVAYSVLVVAKVEKGKSKKLKSVKELLGIT
                     IMERSSFEKNPIDFLEAKGYKEVKKDLIIKLPKYSLFELENGRKRMLASAGELQKGNEL
                     ALPSKYVNFLYLASHYEKLKGSPEDNEQKQLFVEQHKHYLDEIIEQISEFSKRVILADA
                     NLDKVLSAYNKHRDKPIREQAENIIHLFTLTNLGAPAAFKYFDTTIDRKRYTSTKEVLD
                     ATLIHQSITGLYETRIDLSQLGGD"
     CDS             4645..4665
                     /codon_start=1
                     /product="nuclear localization signal of SV40 (simian virus
                     40) large T antigen"
                     /label=SV40 NLS
                     /translation="PKKKRKV"
     misc_feature    4702..5290
                     /label=WPRE
                     /note="woodchuck hepatitis virus posttranscriptional 
                     regulatory element"
     primer_bind     complement(4755..4775)
                     /label=WPRE-R
                     /note="WPRE, reverse primer"
     CDS             complement(5173..5184)
                     /codon_start=1
                     /product="Factor Xa recognition and cleavage site"
                     /label=Factor Xa site
                     /translation="IEGR"
     primer_bind     complement(5293..5309)
                     /label=KS primer
                     /note="common sequencing primer, one of multiple similar 
                     variants"
     primer_bind     complement(5294..5310)
                     /label=pBluescriptKS
                     /note="For pBluescript vector"
     LTR             5815..5995
                     /label=5' LTR (truncated)
                     /note="truncated 5' long terminal repeat (LTR) from HIV-1"
     primer_bind     complement(6021..6038)
                     /label=BGH-rev
                     /note="Bovine growth hormone terminator, reverse primer. 
                     Also called BGH reverse"
     polyA_signal    6027..6251
                     /label=bGH poly(A) signal
                     /note="bovine growth hormone polyadenylation signal"
     rep_origin      6297..6725
                     /direction=RIGHT
                     /label=f1 ori
                     /note="f1 bacteriophage origin of replication; arrow 
                     indicates direction of (+) strand synthesis"
     primer_bind     complement(6384..6403)
                     /label=F1ori-R
                     /note="F1 origin, reverse primer"
     primer_bind     6594..6615
                     /label=F1ori-F
                     /note="F1 origin, forward primer"
     primer_bind     complement(6734..6754)
                     /label=pBABE 3'
                     /note="SV40 enhancer, reverse primer for pBABE vectors"
     promoter        6739..7068
                     /label=SV40 promoter
                     /note="SV40 enhancer and early promoter"
     rep_origin      6919..7054
                     /label=SV40 ori
                     /note="SV40 origin of replication"
     primer_bind     6981..7000
                     /label=SV40pro-F
                     /note="SV40 promoter/origin, forward primer"
     promoter        7116..7163
                     /label=EM7 promoter
                     /note="synthetic bacterial promoter "
     CDS             7182..7556
                     /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    7686..7807
                     /label=SV40 poly(A) signal
                     /note="SV40 polyadenylation signal"
     primer_bind     complement(7723..7742)
                     /label=SV40pA-R
                     /note="SV40 polyA, reverse primer"
     primer_bind     7777..7796
                     /label=EBV-rev
                     /note="SV40 polyA terminator, reverse primer"
     primer_bind     complement(7856..7872)
                     /label=M13 rev
                     /note="common sequencing primer, one of multiple similar 
                     variants"
     primer_bind     complement(7856..7872)
                     /label=M13 Reverse
                     /note="In lacZ gene. Also called M13-rev"
     primer_bind     complement(7869..7891)
                     /label=M13/pUC Reverse
                     /note="In lacZ gene"
     protein_bind    7880..7896
                     /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(7904..7934)
                     /label=lac promoter
                     /note="promoter for the E. coli lac operon"
     protein_bind    7949..7970
                     /label=CAP binding site
                     /bound_moiety="E. coli catabolite activator protein"
                     /note="CAP binding activates transcription in the presence 
                     of cAMP."
     primer_bind     complement(8087..8104)
                     /label=L4440
                     /note="L4440 vector, forward primer"
     rep_origin      complement(8258..8846)
                     /direction=LEFT
                     /label=ori
                     /note="high-copy-number ColE1/pMB1/pBR322/pUC origin of 
                     replication"
     primer_bind     complement(8338..8357)
                     /label=pBR322ori-F
                     /note="pBR322 origin, forward primer"
     CDS             complement(9017..9877)
                     /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     9640..9659
                     /label=Amp-R
                     /note="Ampicillin resistance gene, reverse primer"
     promoter        complement(9878..9982)
                     /gene="bla"
                     /label=AmpR promoter
     primer_bind     complement(10057..10076)
                     /label=pRS-marker
                     /note="pRS vectors, use to sequence yeast selectable 
                     marker"
     enhancer        10248..10627
                     /label=CMV enhancer
                     /note="human cytomegalovirus immediate early enhancer"
     promoter        10629..10827
                     /label=CMV promoter
                     /note="human cytomegalovirus (CMV) immediate early 
                     promoter"
     primer_bind     10782..10802
                     /label=CMV-F
                     /note="Human CMV immediate early promoter, forward primer"
     LTR             10845..11025
                     /label=5' LTR (truncated)
                     /note="truncated 5' long terminal repeat (LTR) from HIV-1"
     misc_feature    11072..11197
                     /label=HIV-1 Psi
                     /note="packaging signal of human immunodeficiency virus 
                     type 1"
     misc_feature    11690..11923
                     /label=RRE
                     /note="The Rev response element (RRE) of HIV-1 allows for 
                     Rev-dependent mRNA export from the nucleus to the 
                     cytoplasm."
     CDS             12108..12152
                     /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    12450..12567
                     /label=cPPT/CTS
                     /note="central polypurine tract and central termination 
                     sequence of HIV-1"
     promoter        12632..425
                     /label=UbC promoter
                     /note="human ubiquitin C promoter"
ORIGIN
        1 gtgccgttgg gcagtgcacc cgtacctttg ggagcgcgcg ccctcgtcgt gtcgtgacgt
       61 cacccgttct gttggcttat aatgcagggt ggggccacct gccggtaggt gtgcggtagg
      121 cttttctccg tcgcaggacg cagggttcgg gcctagggta ggctctcctg aatcgacagg
      181 cgccggacct ctggtgaggg gagggataag tgaggcgtca gtttctttgg tcggttttat
      241 gtacctatct tcttaagtag ctgaagctcc ggttttgaac tatgcgctcg gggttggcga
      301 gtgtgttttg tgaagttttt taggcacctt ttgaaatgta atcatttggg tcaatatgta
      361 attttcagtg ttagactagt aaattgtccg ctaaattctg gccgtttttg gcttttttgt
      421 tagacgaagc ttgggctgca ggtcgactct agaggatccc cgggtaccgg tgccaccatg
      481 tacccatacg atgttccaga ttacgcttcg ccgaagaaaa agcgcaaggt cgaagcgtcc
      541 gacaagaagt acagcatcgg cctggacatc ggcaccaact ctgtgggctg ggccgtgatc
      601 accgacgagt acaaggtgcc cagcaagaaa ttcaaggtgc tgggcaacac cgaccggcac
      661 agcatcaaga agaacctgat cggagccctg ctgttcgaca gcggcgaaac agccgaggcc
      721 acccggctga agagaaccgc cagaagaaga tacaccagac ggaagaaccg gatctgctat
      781 ctgcaagaga tcttcagcaa cgagatggcc aaggtggacg acagcttctt ccacagactg
      841 gaagagtcct tcctggtgga agaggataag aagcacgagc ggcaccccat cttcggcaac
      901 atcgtggacg aggtggccta ccacgagaag taccccacca tctaccacct gagaaagaaa
      961 ctggtggaca gcaccgacaa ggccgacctg cggctgatct atctggccct ggcccacatg
     1021 atcaagttcc ggggccactt cctgatcgag ggcgacctga accccgacaa cagcgacgtg
     1081 gacaagctgt tcatccagct ggtgcagacc tacaaccagc tgttcgagga aaaccccatc
     1141 aacgccagcg gcgtggacgc caaggccatc ctgtctgcca gactgagcaa gagcagacgg
     1201 ctggaaaatc tgatcgccca gctgcccggc gagaagaaga atggcctgtt cggaaacctg
     1261 attgccctga gcctgggcct gacccccaac ttcaagagca acttcgacct ggccgaggat
     1321 gccaaactgc agctgagcaa ggacacctac gacgacgacc tggacaacct gctggcccag
     1381 atcggcgacc agtacgccga cctgtttctg gccgccaaga acctgtccga cgccatcctg
     1441 ctgagcgaca tcctgagagt gaacaccgag atcaccaagg cccccctgag cgcctctatg
     1501 atcaagagat acgacgagca ccaccaggac ctgaccctgc tgaaagctct cgtgcggcag
     1561 cagctgcctg agaagtacaa agagattttc ttcgaccaga gcaagaacgg ctacgccggc
     1621 tacattgacg gcggagccag ccaggaagag ttctacaagt tcatcaagcc catcctggaa
     1681 aagatggacg gcaccgagga actgctcgtg aagctgaaca gagaggacct gctgcggaag
     1741 cagcggacct tcgacaacgg cagcatcccc caccagatcc acctgggaga gctgcacgcc
     1801 attctgcggc ggcaggaaga tttttaccca ttcctgaagg acaaccggga aaagatcgag
     1861 aagatcctga ccttccgcat cccctactac gtgggccctc tggccagggg aaacagcaga
     1921 ttcgcctgga tgaccagaaa gagcgaggaa accatcaccc cctggaactt cgaggaagtg
     1981 gtggacaagg gcgcttccgc ccagagcttc atcgagcgga tgaccaactt cgataagaac
     2041 ctgcccaacg agaaggtgct gcccaagcac agcctgctgt acgagtactt caccgtgtat
     2101 aacgagctga ccaaagtgaa atacgtgacc gagggaatga gaaagcccgc cttcctgagc
     2161 ggcgagcaga aaaaggccat cgtggacctg ctgttcaaga ccaaccggaa agtgaccgtg
     2221 aagcagctga aagaggacta cttcaagaaa atcgagtgct tcgactccgt ggaaatctcc
     2281 ggcgtggaag atcggttcaa cgcctccctg ggcacatacc acgatctgct gaaaattatc
     2341 aaggacaagg acttcctgga caatgaggaa aacgaggaca ttctggaaga tatcgtgctg
     2401 accctgacac tgtttgagga cagagagatg atcgaggaac ggctgaaaac ctatgcccac
     2461 ctgttcgacg acaaagtgat gaagcagctg aagcggcgga gatacaccgg ctggggcagg
     2521 ctgagccgga agctgatcaa cggcatccgg gacaagcagt ccggcaagac aatcctggat
     2581 ttcctgaagt ccgacggctt cgccaacaga aacttcatgc agctgatcca cgacgacagc
     2641 ctgaccttta aagaggacat ccagaaagcc caggtgtccg gccagggcga tagcctgcac
     2701 gagcacattg ccaatctggc cggcagcccc gccattaaga agggcatcct gcagacagtg
     2761 aaggtggtgg acgagctcgt gaaagtgatg ggccggcaca agcccgagaa catcgtgatc
     2821 gaaatggcca gagagaacca gaccacccag aagggacaga agaacagccg cgagagaatg
     2881 aagcggatcg aagagggcat caaagagctg ggcagccaga tcctgaaaga acaccccgtg
     2941 gaaaacaccc agctgcagaa cgagaagctg tacctgtact acctgcagaa tgggcgggat
     3001 atgtacgtgg accaggaact ggacatcaac cggctgtccg actacgatgt ggaccatatc
     3061 gtgcctcaga gctttctgaa ggacgactcc atcgacaaca aggtgctgac cagaagcgac
     3121 aagaaccggg gcaagagcga caacgtgccc tccgaagagg tcgtgaagaa gatgaagaac
     3181 tactggcggc agctgctgaa cgccaagctg attacccaga gaaagttcga caatctgacc
     3241 aaggccgaga gaggcggcct gagcgaactg gataaggccg gcttcatcaa gagacagctg
     3301 gtggaaaccc ggcagatcac aaagcacgtg gcacagatcc tggactcccg gatgaacact
     3361 aagtacgacg agaatgacaa gctgatccgg gaagtgaaag tgatcaccct gaagtccaag
     3421 ctggtgtccg atttccggaa ggatttccag ttttacaaag tgcgcgagat caacaactac
     3481 caccacgccc acgacgccta cctgaacgcc gtcgtgggaa ccgccctgat caaaaagtac
     3541 cctaagctgg aaagcgagtt cgtgtacggc gactacaagg tgtacgacgt gcggaagatg
     3601 atcgccaaga gcgagcagga aatcggcaag gctaccgcca agtacttctt ctacagcaac
     3661 atcatgaact ttttcaagac cgagattacc ctggccaacg gcgagatccg gaagcggcct
     3721 ctgatcgaga caaacggcga aaccggggag atcgtgtggg ataagggccg ggattttgcc
     3781 accgtgcgga aagtgctgag catgccccaa gtgaatatcg tgaaaaagac cgaggtgcag
     3841 acaggcggct tcagcaaaga gtctatcctg cccaagagga acagcgataa gctgatcgcc
     3901 agaaagaagg actgggaccc taagaagtac ggcggcttcg acagccccac cgtggcctat
     3961 tctgtgctgg tggtggccaa agtggaaaag ggcaagtcca agaaactgaa gagtgtgaaa
     4021 gagctgctgg ggatcaccat catggaaaga agcagcttcg agaagaatcc catcgacttt
     4081 ctggaagcca agggctacaa agaagtgaaa aaggacctga tcatcaagct gcctaagtac
     4141 tccctgttcg agctggaaaa cggccggaag agaatgctgg cctctgccgg cgaactgcag
     4201 aagggaaacg aactggccct gccctccaaa tatgtgaact tcctgtacct ggccagccac
     4261 tatgagaagc tgaagggctc ccccgaggat aatgagcaga aacagctgtt tgtggaacag
     4321 cacaagcact acctggacga gatcatcgag cagatcagcg agttctccaa gagagtgatc
     4381 ctggccgacg ctaatctgga caaagtgctg tccgcctaca acaagcaccg ggataagccc
     4441 atcagagagc aggccgagaa tatcatccac ctgtttaccc tgaccaatct gggagcccct
     4501 gccgccttca agtactttga caccaccatc gaccggaaga ggtacaccag caccaaagag
     4561 gtgctggacg ccaccctgat ccaccagagc atcaccggcc tgtacgagac acggatcgac
     4621 ctgtctcagc tgggaggcga cagccccaag aagaagagaa aggtggaggc cagctaagaa
     4681 ttcgatatca agcttatcga taatcaacct ctggattaca aaatttgtga aagattgact
     4741 ggtattctta actatgttgc tccttttacg ctatgtggat acgctgcttt aatgcctttg
     4801 tatcatgcta ttgcttcccg tatggctttc attttctcct ccttgtataa atcctggttg
     4861 ctgtctcttt atgaggagtt gtggcccgtt gtcaggcaac gtggcgtggt gtgcactgtg
     4921 tttgctgacg caacccccac tggttggggc attgccacca cctgtcagct cctttccggg
     4981 actttcgctt tccccctccc tattgccacg gcggaactca tcgccgcctg ccttgcccgc
     5041 tgctggacag gggctcggct gttgggcact gacaattccg tggtgttgtc ggggaaatca
     5101 tcgtcctttc cttggctgct cgcctgtgtt gccacctgga ttctgcgcgg gacgtccttc
     5161 tgctacgtcc cttcggccct caatccagcg gaccttcctt cccgcggcct gctgccggct
     5221 ctgcggcctc ttccgcgtct tcgccttcgc cctcagacga gtcggatctc cctttgggcc
     5281 gcctccccgc atcgataccg tcgacctcga gacctagaaa aacatggagc aatcacaagt
     5341 agcaatacag cagctaccaa tgctgattgt gcctggctag aagcacaaga ggaggaggag
     5401 gtgggttttc cagtcacacc tcaggtacct ttaagaccaa tgacttacaa ggcagctgta
     5461 gatcttagcc actttttaaa agaaaagggg ggactggaag ggctaattca ctcccaacga
     5521 agacaagata tccttgatct gtggatctac cacacacaag gctacttccc tgattggcag
     5581 aactacacac cagggccagg gatcagatat ccactgacct ttggatggtg ctacaagcta
     5641 gtaccagttg agcaagagaa ggtagaagaa gccaatgaag gagagaacac ccgcttgtta
     5701 caccctgtga gcctgcatgg gatggatgac ccggagagag aagtattaga gtggaggttt
     5761 gacagccgcc tagcatttca tcacatggcc cgagagctgc atccggactg tactgggtct
     5821 ctctggttag accagatctg agcctgggag ctctctggct aactagggaa cccactgctt
     5881 aagcctcaat aaagcttgcc ttgagtgctt caagtagtgt gtgcccgtct gttgtgtgac
     5941 tctggtaact agagatccct cagacccttt tagtcagtgt ggaaaatctc tagcagggcc
     6001 cgtttaaacc cgctgatcag cctcgactgt gccttctagt tgccagccat ctgttgtttg
     6061 cccctccccc gtgccttcct tgaccctgga aggtgccact cccactgtcc tttcctaata
     6121 aaatgaggaa attgcatcgc attgtctgag taggtgtcat tctattctgg ggggtggggt
     6181 ggggcaggac agcaaggggg aggattggga agacaatagc aggcatgctg gggatgcggt
     6241 gggctctatg gcttctgagg cggaaagaac cagctggggc tctagggggt atccccacgc
     6301 gccctgtagc ggcgcattaa gcgcggcggg tgtggtggtt acgcgcagcg tgaccgctac
     6361 acttgccagc gccctagcgc ccgctccttt cgctttcttc ccttcctttc tcgccacgtt
     6421 cgccggcttt ccccgtcaag ctctaaatcg ggggctccct ttagggttcc gatttagtgc
     6481 tttacggcac ctcgacccca aaaaacttga ttagggtgat ggttcacgta gtgggccatc
     6541 gccctgatag acggtttttc gccctttgac gttggagtcc acgttcttta atagtggact
     6601 cttgttccaa actggaacaa cactcaaccc tatctcggtc tattcttttg atttataagg
     6661 gattttgccg atttcggcct attggttaaa aaatgagctg atttaacaaa aatttaacgc
     6721 gaattaattc tgtggaatgt gtgtcagtta gggtgtggaa agtccccagg ctccccagca
     6781 ggcagaagta tgcaaagcat gcatctcaat tagtcagcaa ccaggtgtgg aaagtcccca
     6841 ggctccccag caggcagaag tatgcaaagc atgcatctca attagtcagc aaccatagtc
     6901 ccgcccctaa ctccgcccat cccgccccta actccgccca gttccgccca ttctccgccc
     6961 catggctgac taattttttt tatttatgca gaggccgagg ccgcctctgc ctctgagcta
     7021 ttccagaagt agtgaggagg cttttttgga ggcctaggct tttgcaaaaa gctcccggga
     7081 gcttgtatat ccattttcgg atctgatcag cacgtgttga caattaatca tcggcatagt
     7141 atatcggcat agtataatac gacaaggtga ggaactaaac catggccaag ttgaccagtg
     7201 ccgttccggt gctcaccgcg cgcgacgtcg ccggagcggt cgagttctgg accgaccggc
     7261 tcgggttctc ccgggacttc gtggaggacg acttcgccgg tgtggtccgg gacgacgtga
     7321 ccctgttcat cagcgcggtc caggaccagg tggtgccgga caacaccctg gcctgggtgt
     7381 gggtgcgcgg cctggacgag ctgtacgccg agtggtcgga ggtcgtgtcc acgaacttcc
     7441 gggacgcctc cgggccggcc atgaccgaga tcggcgagca gccgtggggg cgggagttcg
     7501 ccctgcgcga cccggccggc aactgcgtgc acttcgtggc cgaggagcag gactgacacg
     7561 tgctacgaga tttcgattcc accgccgcct tctatgaaag gttgggcttc ggaatcgttt
     7621 tccgggacgc cggctggatg atcctccagc gcggggatct catgctggag ttcttcgccc
     7681 accccaactt gtttattgca gcttataatg gttacaaata aagcaatagc atcacaaatt
     7741 tcacaaataa agcatttttt tcactgcatt ctagttgtgg tttgtccaaa ctcatcaatg
     7801 tatcttatca tgtctgtata ccgtcgacct ctagctagag cttggcgtaa tcatggtcat
     7861 agctgtttcc tgtgtgaaat tgttatccgc tcacaattcc acacaacata cgagccggaa
     7921 gcataaagtg taaagcctgg ggtgcctaat gagtgagcta actcacatta attgcgttgc
     7981 gctcactgcc cgctttccag tcgggaaacc tgtcgtgcca gctgcattaa tgaatcggcc
     8041 aacgcgcggg gagaggcggt ttgcgtattg ggcgctcttc cgcttcctcg ctcactgact
     8101 cgctgcgctc ggtcgttcgg ctgcggcgag cggtatcagc tcactcaaag gcggtaatac
     8161 ggttatccac agaatcaggg gataacgcag gaaagaacat gtgagcaaaa ggccagcaaa
     8221 aggccaggaa ccgtaaaaag gccgcgttgc tggcgttttt ccataggctc cgcccccctg
     8281 acgagcatca caaaaatcga cgctcaagtc agaggtggcg aaacccgaca ggactataaa
     8341 gataccaggc gtttccccct ggaagctccc tcgtgcgctc tcctgttccg accctgccgc
     8401 ttaccggata cctgtccgcc tttctccctt cgggaagcgt ggcgctttct catagctcac
     8461 gctgtaggta tctcagttcg gtgtaggtcg ttcgctccaa gctgggctgt gtgcacgaac
     8521 cccccgttca gcccgaccgc tgcgccttat ccggtaacta tcgtcttgag tccaacccgg
     8581 taagacacga cttatcgcca ctggcagcag ccactggtaa caggattagc agagcgaggt
     8641 atgtaggcgg tgctacagag ttcttgaagt ggtggcctaa ctacggctac actagaagaa
     8701 cagtatttgg tatctgcgct ctgctgaagc cagttacctt cggaaaaaga gttggtagct
     8761 cttgatccgg caaacaaacc accgctggta gcggtggttt ttttgtttgc aagcagcaga
     8821 ttacgcgcag aaaaaaagga tctcaagaag atcctttgat cttttctacg gggtctgacg
     8881 ctcagtggaa cgaaaactca cgttaaggga ttttggtcat gagattatca aaaaggatct
     8941 tcacctagat ccttttaaat taaaaatgaa gttttaaatc aatctaaagt atatatgagt
     9001 aaacttggtc tgacagttac caatgcttaa tcagtgaggc acctatctca gcgatctgtc
     9061 tatttcgttc atccatagtt gcctgactcc ccgtcgtgta gataactacg atacgggagg
     9121 gcttaccatc tggccccagt gctgcaatga taccgcgaga cccacgctca ccggctccag
     9181 atttatcagc aataaaccag ccagccggaa gggccgagcg cagaagtggt cctgcaactt
     9241 tatccgcctc catccagtct attaattgtt gccgggaagc tagagtaagt agttcgccag
     9301 ttaatagttt gcgcaacgtt gttgccattg ctacaggcat cgtggtgtca cgctcgtcgt
     9361 ttggtatggc ttcattcagc tccggttccc aacgatcaag gcgagttaca tgatccccca
     9421 tgttgtgcaa aaaagcggtt agctccttcg gtcctccgat cgttgtcaga agtaagttgg
     9481 ccgcagtgtt atcactcatg gttatggcag cactgcataa ttctcttact gtcatgccat
     9541 ccgtaagatg cttttctgtg actggtgagt actcaaccaa gtcattctga gaatagtgta
     9601 tgcggcgacc gagttgctct tgcccggcgt caatacggga taataccgcg ccacatagca
     9661 gaactttaaa agtgctcatc attggaaaac gttcttcggg gcgaaaactc tcaaggatct
     9721 taccgctgtt gagatccagt tcgatgtaac ccactcgtgc acccaactga tcttcagcat
     9781 cttttacttt caccagcgtt tctgggtgag caaaaacagg aaggcaaaat gccgcaaaaa
     9841 agggaataag ggcgacacgg aaatgttgaa tactcatact cttccttttt caatattatt
     9901 gaagcattta tcagggttat tgtctcatga gcggatacat atttgaatgt atttagaaaa
     9961 ataaacaaat aggggttccg cgcacatttc cccgaaaagt gccacctgac gtcgacggat
    10021 cgggagatct cccgatcccc tatggtgcac tctcagtaca atctgctctg atgccgcata
    10081 gttaagccag tatctgctcc ctgcttgtgt gttggaggtc gctgagtagt gcgcgagcaa
    10141 aatttaagct acaacaaggc aaggcttgac cgacaattgc atgaagaatc tgcttagggt
    10201 taggcgtttt gcgctgcttc gcgatgtacg ggccagatat acgcgttgac attgattatt
    10261 gactagttat taatagtaat caattacggg gtcattagtt catagcccat atatggagtt
    10321 ccgcgttaca taacttacgg taaatggccc gcctggctga ccgcccaacg acccccgccc
    10381 attgacgtca ataatgacgt atgttcccat agtaacgcca atagggactt tccattgacg
    10441 tcaatgggtg gagtatttac ggtaaactgc ccacttggca gtacatcaag tgtatcatat
    10501 gccaagtacg ccccctattg acgtcaatga cggtaaatgg cccgcctggc attatgccca
    10561 gtacatgacc ttatgggact ttcctacttg gcagtacatc tacgtattag tcatcgctat
    10621 taccatggtg atgcggtttt ggcagtacat caatgggcgt ggatagcggt ttgactcacg
    10681 gggatttcca agtctccacc ccattgacgt caatgggagt ttgttttggc accaaaatca
    10741 acgggacttt ccaaaatgtc gtaacaactc cgccccattg acgcaaatgg gcggtaggcg
    10801 tgtacggtgg gaggtctata taagcagcgc gttttgcctg tactgggtct ctctggttag
    10861 accagatctg agcctgggag ctctctggct aactagggaa cccactgctt aagcctcaat
    10921 aaagcttgcc ttgagtgctt caagtagtgt gtgcccgtct gttgtgtgac tctggtaact
    10981 agagatccct cagacccttt tagtcagtgt ggaaaatctc tagcagtggc gcccgaacag
    11041 ggacttgaaa gcgaaaggga aaccagagga gctctctcga cgcaggactc ggcttgctga
    11101 agcgcgcacg gcaagaggcg aggggcggcg actggtgagt acgccaaaaa ttttgactag
    11161 cggaggctag aaggagagag atgggtgcga gagcgtcagt attaagcggg ggagaattag
    11221 atcgcgatgg gaaaaaattc ggttaaggcc agggggaaag aaaaaatata aattaaaaca
    11281 tatagtatgg gcaagcaggg agctagaacg attcgcagtt aatcctggcc tgttagaaac
    11341 atcagaaggc tgtagacaaa tactgggaca gctacaacca tcccttcaga caggatcaga
    11401 agaacttaga tcattatata atacagtagc aaccctctat tgtgtgcatc aaaggataga
    11461 gataaaagac accaaggaag ctttagacaa gatagaggaa gagcaaaaca aaagtaagac
    11521 caccgcacag caagcggccg ctgatcttca gacctggagg aggagatatg agggacaatt
    11581 ggagaagtga attatataaa tataaagtag taaaaattga accattagga gtagcaccca
    11641 ccaaggcaaa gagaagagtg gtgcagagag aaaaaagagc agtgggaata ggagctttgt
    11701 tccttgggtt cttgggagca gcaggaagca ctatgggcgc agcgtcaatg acgctgacgg
    11761 tacaggccag acaattattg tctggtatag tgcagcagca gaacaatttg ctgagggcta
    11821 ttgaggcgca acagcatctg ttgcaactca cagtctgggg catcaagcag ctccaggcaa
    11881 gaatcctggc tgtggaaaga tacctaaagg atcaacagct cctggggatt tggggttgct
    11941 ctggaaaact catttgcacc actgctgtgc cttggaatgc tagttggagt aataaatctc
    12001 tggaacagat ttggaatcac acgacctgga tggagtggga cagagaaatt aacaattaca
    12061 caagcttaat acactcctta attgaagaat cgcaaaacca gcaagaaaag aatgaacaag
    12121 aattattgga attagataaa tgggcaagtt tgtggaattg gtttaacata acaaattggc
    12181 tgtggtatat aaaattattc ataatgatag taggaggctt ggtaggttta agaatagttt
    12241 ttgctgtact ttctatagtg aatagagtta ggcagggata ttcaccatta tcgtttcaga
    12301 cccacctccc aaccccgagg ggacccgaca ggcccgaagg aatagaagaa gaaggtggag
    12361 agagagacag agacagatcc attcgattag tgaacggatc ggcactgcgt gcgccaattc
    12421 tgcagacaaa tggcagtatt catccacaat tttaaaagaa aaggggggat tggggggtac
    12481 agtgcagggg aaagaatagt agacataata gcaacagaca tacaaactaa agaattacaa
    12541 aaacaaatta caaaaattca aaattttcgg gtttattaca gggacagcag agatccagtt
    12601 tggttaatta acccgtgtcg gctccagatc tggcctccgc gccgggtttt ggcgcctccc
    12661 gcgggcgccc ccctcctcac ggcgagcgct gccacgtcag acgaagggcg cagcgagcgt
    12721 cctgatcctt ccgcccggac gctcaggaca gcggcccgct gctcataaga ctcggcctta
    12781 gaaccccagt atcagcagaa ggacatttta ggacgggact tgggtgactc tagggcactg
    12841 gttttctttc cagagagcgg aacaggcgag gaaaagtagt cccttctcgg cgattctgcg
    12901 gagggatctc cgtggggcgg tgaacgccga tgattatata aggacgcgcc gggtgtggca
    12961 cagctagttc cgtcgcagcc gggatttggg tcgcggttct tgtttgtgga tcgctgtgat
    13021 cgtcacttgg tgagtagcgg gctgctgggc tggccggggc tttcgtggcc gccgggccgc
    13081 tcggtgggac ggaagcgtgt ggagagaccg ccaagggctg tagtctgggt ccgcgagcaa
    13141 ggttgccctg aactgggggt tggggggagc gcagcaaaat ggcggctgtt cccgagtctt
    13201 gaatggaaga cgcttgtgag gcgggctgtg aggtcgttga aacaaggtgg ggggcatggt
    13261 gggcggcaag aacccaaggt cttgaggcct tcgctaatgc gggaaagctc ttattcgggt
    13321 gagatgggct ggggcaccat ctggggaccc tgacgtgaag tttgtcactg actggagaac
    13381 tcggtttgtc gtctgttgcg ggggcggcag ttatggcg
//