LOCUS       sequence-433154-       13355 bp    DNA     circular SYN 15-APR-2025
DEFINITION  A knockout vector for dog Merlin..
ACCESSION   .
VERSION     .
KEYWORDS    .
SOURCE      synthetic DNA construct
  ORGANISM  synthetic DNA construct
REFERENCE   1  (bases 1 to 13355)
  TITLE     Collective cell migration is coordinated by front-biased activation 
            of the feed-forward loop consisting of Ras, Rab5, and Rac1.
  JOURNAL   Unpublished
REFERENCE   2  (bases 1 to 13355)
  AUTHORS   .
  TITLE     Direct Submission
  JOURNAL   Exported Apr 15, 2025 from SnapGene Server 8.0.1
            https://www.snapgene.com
COMMENT     SGRef: number: 1; type: "Journal Article"; journalName: 
            "Unpublished"
COMMENT     Sequence Label: lentiCRISPRv2-bleo-Merlin(dog)gRNA2
FEATURES             Location/Qualifiers
     source          1..13355
                     /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"
     misc_RNA        2878..2953
                     /label=gRNA scaffold
                     /note="guide RNA scaffold for the Streptococcus pyogenes 
                     CRISPR/Cas9 system"
     promoter        3015..3226
                     /label=EF-1-alpha core promoter
                     /note="core promoter for human elongation factor 
                     EF-1-alpha"
     regulatory      3245..3254
                     /label=Kozak sequence
                     /note="vertebrate consensus sequence for strong initiation 
                     of translation (Kozak, 1987)"
                     /regulatory_class="other"
     CDS             3251..7354
                     /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="MDKKYSIGLDIGTNSVGWAVITDEYKVPSKKFKVLGNTDRHSIKK
                     NLIGALLFDSGETAEATRLKRTARRRYTRRKNRICYLQEIFSNEMAKVDDSFFHRLEES
                     FLVEEDKKHERHPIFGNIVDEVAYHEKYPTIYHLRKKLVDSTDKADLRLIYLALAHMIK
                     FRGHFLIEGDLNPDNSDVDKLFIQLVQTYNQLFEENPINASGVDAKAILSARLSKSRRL
                     ENLIAQLPGEKKNGLFGNLIALSLGLTPNFKSNFDLAEDAKLQLSKDTYDDDLDNLLAQ
                     IGDQYADLFLAAKNLSDAILLSDILRVNTEITKAPLSASMIKRYDEHHQDLTLLKALVR
                     QQLPEKYKEIFFDQSKNGYAGYIDGGASQEEFYKFIKPILEKMDGTEELLVKLNREDLL
                     RKQRTFDNGSIPHQIHLGELHAILRRQEDFYPFLKDNREKIEKILTFRIPYYVGPLARG
                     NSRFAWMTRKSEETITPWNFEEVVDKGASAQSFIERMTNFDKNLPNEKVLPKHSLLYEY
                     FTVYNELTKVKYVTEGMRKPAFLSGEQKKAIVDLLFKTNRKVTVKQLKEDYFKKIECFD
                     SVEISGVEDRFNASLGTYHDLLKIIKDKDFLDNEENEDILEDIVLTLTLFEDREMIEER
                     LKTYAHLFDDKVMKQLKRRRYTGWGRLSRKLINGIRDKQSGKTILDFLKSDGFANRNFM
                     QLIHDDSLTFKEDIQKAQVSGQGDSLHEHIANLAGSPAIKKGILQTVKVVDELVKVMGR
                     HKPENIVIEMARENQTTQKGQKNSRERMKRIEEGIKELGSQILKEHPVENTQLQNEKLY
                     LYYLQNGRDMYVDQELDINRLSDYDVDHIVPQSFLKDDSIDNKVLTRSDKNRGKSDNVP
                     SEEVVKKMKNYWRQLLNAKLITQRKFDNLTKAERGGLSELDKAGFIKRQLVETRQITKH
                     VAQILDSRMNTKYDENDKLIREVKVITLKSKLVSDFRKDFQFYKVREINNYHHAHDAYL
                     NAVVGTALIKKYPKLESEFVYGDYKVYDVRKMIAKSEQEIGKATAKYFFYSNIMNFFKT
                     EITLANGEIRKRPLIETNGETGEIVWDKGRDFATVRKVLSMPQVNIVKKTEVQTGGFSK
                     ESILPKRNSDKLIARKKDWDPKKYGGFDSPTVAYSVLVVAKVEKGKSKKLKSVKELLGI
                     TIMERSSFEKNPIDFLEAKGYKEVKKDLIIKLPKYSLFELENGRKRMLASAGELQKGNE
                     LALPSKYVNFLYLASHYEKLKGSPEDNEQKQLFVEQHKHYLDEIIEQISEFSKRVILAD
                     ANLDKVLSAYNKHRDKPIREQAENIIHLFTLTNLGAPAAFKYFDTTIDRKRYTSTKEVL
                     DATLIHQSITGLYETRIDLSQLGGD"
     CDS             3251..7354
                     /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="MDKKYSIGLDIGTNSVGWAVITDEYKVPSKKFKVLGNTDRHSIKK
                     NLIGALLFDSGETAEATRLKRTARRRYTRRKNRICYLQEIFSNEMAKVDDSFFHRLEES
                     FLVEEDKKHERHPIFGNIVDEVAYHEKYPTIYHLRKKLVDSTDKADLRLIYLALAHMIK
                     FRGHFLIEGDLNPDNSDVDKLFIQLVQTYNQLFEENPINASGVDAKAILSARLSKSRRL
                     ENLIAQLPGEKKNGLFGNLIALSLGLTPNFKSNFDLAEDAKLQLSKDTYDDDLDNLLAQ
                     IGDQYADLFLAAKNLSDAILLSDILRVNTEITKAPLSASMIKRYDEHHQDLTLLKALVR
                     QQLPEKYKEIFFDQSKNGYAGYIDGGASQEEFYKFIKPILEKMDGTEELLVKLNREDLL
                     RKQRTFDNGSIPHQIHLGELHAILRRQEDFYPFLKDNREKIEKILTFRIPYYVGPLARG
                     NSRFAWMTRKSEETITPWNFEEVVDKGASAQSFIERMTNFDKNLPNEKVLPKHSLLYEY
                     FTVYNELTKVKYVTEGMRKPAFLSGEQKKAIVDLLFKTNRKVTVKQLKEDYFKKIECFD
                     SVEISGVEDRFNASLGTYHDLLKIIKDKDFLDNEENEDILEDIVLTLTLFEDREMIEER
                     LKTYAHLFDDKVMKQLKRRRYTGWGRLSRKLINGIRDKQSGKTILDFLKSDGFANRNFM
                     QLIHDDSLTFKEDIQKAQVSGQGDSLHEHIANLAGSPAIKKGILQTVKVVDELVKVMGR
                     HKPENIVIEMARENQTTQKGQKNSRERMKRIEEGIKELGSQILKEHPVENTQLQNEKLY
                     LYYLQNGRDMYVDQELDINRLSDYDVDHIVPQSFLKDDSIDNKVLTRSDKNRGKSDNVP
                     SEEVVKKMKNYWRQLLNAKLITQRKFDNLTKAERGGLSELDKAGFIKRQLVETRQITKH
                     VAQILDSRMNTKYDENDKLIREVKVITLKSKLVSDFRKDFQFYKVREINNYHHAHDAYL
                     NAVVGTALIKKYPKLESEFVYGDYKVYDVRKMIAKSEQEIGKATAKYFFYSNIMNFFKT
                     EITLANGEIRKRPLIETNGETGEIVWDKGRDFATVRKVLSMPQVNIVKKTEVQTGGFSK
                     ESILPKRNSDKLIARKKDWDPKKYGGFDSPTVAYSVLVVAKVEKGKSKKLKSVKELLGI
                     TIMERSSFEKNPIDFLEAKGYKEVKKDLIIKLPKYSLFELENGRKRMLASAGELQKGNE
                     LALPSKYVNFLYLASHYEKLKGSPEDNEQKQLFVEQHKHYLDEIIEQISEFSKRVILAD
                     ANLDKVLSAYNKHRDKPIREQAENIIHLFTLTNLGAPAAFKYFDTTIDRKRYTSTKEVL
                     DATLIHQSITGLYETRIDLSQLGGD"
     CDS             7355..7402
                     /codon_start=1
                     /product="bipartite nuclear localization signal from 
                     nucleoplasmin"
                     /label=nucleoplasmin NLS
                     /translation="KRPAATKKAGQAKKKK"
     CDS             7403..7426
                     /codon_start=1
                     /product="FLAG(R) epitope tag, followed by an enterokinase 
                     cleavage site"
                     /label=FLAG
                     /translation="DYKDDDDK"
     misc_feature    7505..8054
                     /label=IRES
                     /note="internal ribosome entry site (IRES) of the 
                     encephalomyocarditis virus (EMCV)"
     primer_bind     complement(7649..7666)
                     /label=IRES reverse
                     /note="IRES internal ribosome entry site, reverse primer. 
                     Also called pCDH-rev"
     primer_bind     7876..7895
                     /label=IRES-F
                     /note="IRES internal ribosome entry site, forward primer"
     CDS             8057..8431
                     /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"
     misc_feature    8447..9035
                     /label=WPRE
                     /note="woodchuck hepatitis virus posttranscriptional 
                     regulatory element"
     primer_bind     complement(8500..8520)
                     /label=WPRE-R
                     /note="WPRE, reverse primer"
     CDS             complement(8918..8929)
                     /codon_start=1
                     /product="Factor Xa recognition and cleavage site"
                     /label=Factor Xa site
                     /translation="IEGR"
     LTR             9107..9340
                     /label=3' LTR (Delta-U3)
                     /note="self-inactivating 3' long terminal repeat (LTR) from
                     HIV-1"
     primer_bind     complement(9366..9383)
                     /label=BGH-rev
                     /note="Bovine growth hormone terminator, reverse primer. 
                     Also called BGH reverse"
     polyA_signal    9372..9596
                     /label=bGH poly(A) signal
                     /note="bovine growth hormone polyadenylation signal"
     rep_origin      9642..10070
                     /direction=RIGHT
                     /label=f1 ori
                     /note="f1 bacteriophage origin of replication; arrow 
                     indicates direction of (+) strand synthesis"
     primer_bind     complement(9729..9748)
                     /label=F1ori-R
                     /note="F1 origin, reverse primer"
     primer_bind     9939..9960
                     /label=F1ori-F
                     /note="F1 origin, forward primer"
     primer_bind     complement(10079..10099)
                     /label=pBABE 3'
                     /note="SV40 enhancer, reverse primer for pBABE vectors"
     promoter        10084..10413
                     /label=SV40 promoter
                     /note="SV40 enhancer and early promoter"
     rep_origin      10264..10399
                     /label=SV40 ori
                     /note="SV40 origin of replication"
     primer_bind     10326..10345
                     /label=SV40pro-F
                     /note="SV40 promoter/origin, forward primer"
     promoter        10461..10508
                     /label=EM7 promoter
                     /note="synthetic bacterial promoter "
     CDS             10527..10901
                     /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    11031..11152
                     /label=SV40 poly(A) signal
                     /note="SV40 polyadenylation signal"
     primer_bind     complement(11068..11087)
                     /label=SV40pA-R
                     /note="SV40 polyA, reverse primer"
     primer_bind     11122..11141
                     /label=EBV-rev
                     /note="SV40 polyA terminator, reverse primer"
     primer_bind     complement(11201..11217)
                     /label=M13 rev
                     /note="common sequencing primer, one of multiple similar 
                     variants"
     primer_bind     complement(11201..11217)
                     /label=M13 Reverse
                     /note="In lacZ gene. Also called M13-rev"
     primer_bind     complement(11214..11236)
                     /label=M13/pUC Reverse
                     /note="In lacZ gene"
     protein_bind    11225..11241
                     /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(11249..11255,11256..11273,11274..11279))
                     /label=lac promoter
                     /note="promoter for the E. coli lac operon"
     protein_bind    11294..11315
                     /label=CAP binding site
                     /bound_moiety="E. coli catabolite activator protein"
                     /note="CAP binding activates transcription in the presence 
                     of cAMP."
     primer_bind     complement(11432..11449)
                     /label=L4440
                     /note="L4440 vector, forward primer"
     rep_origin      complement(11603..12191)
                     /direction=LEFT
                     /label=ori
                     /note="high-copy-number ColE1/pMB1/pBR322/pUC origin of 
                     replication"
     primer_bind     complement(11683..11702)
                     /label=pBR322ori-F
                     /note="pBR322 origin, forward primer"
     CDS             complement(join(12362..13153,13154..13222))
                     /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     12985..13004
                     /label=Amp-R
                     /note="Ampicillin resistance gene, reverse primer"
     promoter        complement(13223..13327)
                     /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 aacaccgagt tgattccgta catttccgtt
     2881 ttagagctag aaatagcaag ttaaaataag gctagtccgt tatcaacttg aaaaagtggc
     2941 accgagtcgg tgcttttttg aattcgctag ctaggtcttg aaaggagtgg gaattggctc
     3001 cggtgcccgt cagtgggcag agcgcacatc gcccacagtc cccgagaagt tggggggagg
     3061 ggtcggcaat tgatccggtg cctagagaag gtggcgcggg gtaaactggg aaagtgatgt
     3121 cgtgtactgg ctccgccttt ttcccgaggg tgggggagaa ccgtatataa gtgcagtagt
     3181 cgccgtgaac gttctttttc gcaacgggtt tgccgccaga acacaggacc ggttctagag
     3241 cgctgccacc atggacaaga agtacagcat cggcctggac atcggcacca actctgtggg
     3301 ctgggccgtg atcaccgacg agtacaaggt gcccagcaag aaattcaagg tgctgggcaa
     3361 caccgaccgg cacagcatca agaagaacct gatcggagcc ctgctgttcg acagcggcga
     3421 aacagccgag gccacccggc tgaagagaac cgccagaaga agatacacca gacggaagaa
     3481 ccggatctgc tatctgcaag agatcttcag caacgagatg gccaaggtgg acgacagctt
     3541 cttccacaga ctggaagagt ccttcctggt ggaagaggat aagaagcacg agcggcaccc
     3601 catcttcggc aacatcgtgg acgaggtggc ctaccacgag aagtacccca ccatctacca
     3661 cctgagaaag aaactggtgg acagcaccga caaggccgac ctgcggctga tctatctggc
     3721 cctggcccac atgatcaagt tccggggcca cttcctgatc gagggcgacc tgaaccccga
     3781 caacagcgac gtggacaagc tgttcatcca gctggtgcag acctacaacc agctgttcga
     3841 ggaaaacccc atcaacgcca gcggcgtgga cgccaaggcc atcctgtctg ccagactgag
     3901 caagagcaga cggctggaaa atctgatcgc ccagctgccc ggcgagaaga agaatggcct
     3961 gttcggaaac ctgattgccc tgagcctggg cctgaccccc aacttcaaga gcaacttcga
     4021 cctggccgag gatgccaaac tgcagctgag caaggacacc tacgacgacg acctggacaa
     4081 cctgctggcc cagatcggcg accagtacgc cgacctgttt ctggccgcca agaacctgtc
     4141 cgacgccatc ctgctgagcg acatcctgag agtgaacacc gagatcacca aggcccccct
     4201 gagcgcctct atgatcaaga gatacgacga gcaccaccag gacctgaccc tgctgaaagc
     4261 tctcgtgcgg cagcagctgc ctgagaagta caaagagatt ttcttcgacc agagcaagaa
     4321 cggctacgcc ggctacattg acggcggagc cagccaggaa gagttctaca agttcatcaa
     4381 gcccatcctg gaaaagatgg acggcaccga ggaactgctc gtgaagctga acagagagga
     4441 cctgctgcgg aagcagcgga ccttcgacaa cggcagcatc ccccaccaga tccacctggg
     4501 agagctgcac gccattctgc ggcggcagga agatttttac ccattcctga aggacaaccg
     4561 ggaaaagatc gagaagatcc tgaccttccg catcccctac tacgtgggcc ctctggccag
     4621 gggaaacagc agattcgcct ggatgaccag aaagagcgag gaaaccatca ccccctggaa
     4681 cttcgaggaa gtggtggaca agggcgcttc cgcccagagc ttcatcgagc ggatgaccaa
     4741 cttcgataag aacctgccca acgagaaggt gctgcccaag cacagcctgc tgtacgagta
     4801 cttcaccgtg tataacgagc tgaccaaagt gaaatacgtg accgagggaa tgagaaagcc
     4861 cgccttcctg agcggcgagc agaaaaaggc catcgtggac ctgctgttca agaccaaccg
     4921 gaaagtgacc gtgaagcagc tgaaagagga ctacttcaag aaaatcgagt gcttcgactc
     4981 cgtggaaatc tccggcgtgg aagatcggtt caacgcctcc ctgggcacat accacgatct
     5041 gctgaaaatt atcaaggaca aggacttcct ggacaatgag gaaaacgagg acattctgga
     5101 agatatcgtg ctgaccctga cactgtttga ggacagagag atgatcgagg aacggctgaa
     5161 aacctatgcc cacctgttcg acgacaaagt gatgaagcag ctgaagcggc ggagatacac
     5221 cggctggggc aggctgagcc ggaagctgat caacggcatc cgggacaagc agtccggcaa
     5281 gacaatcctg gatttcctga agtccgacgg cttcgccaac agaaacttca tgcagctgat
     5341 ccacgacgac agcctgacct ttaaagagga catccagaaa gcccaggtgt ccggccaggg
     5401 cgatagcctg cacgagcaca ttgccaatct ggccggcagc cccgccatta agaagggcat
     5461 cctgcagaca gtgaaggtgg tggacgagct cgtgaaagtg atgggccggc acaagcccga
     5521 gaacatcgtg atcgaaatgg ccagagagaa ccagaccacc cagaagggac agaagaacag
     5581 ccgcgagaga atgaagcgga tcgaagaggg catcaaagag ctgggcagcc agatcctgaa
     5641 agaacacccc gtggaaaaca cccagctgca gaacgagaag ctgtacctgt actacctgca
     5701 gaatgggcgg gatatgtacg tggaccagga actggacatc aaccggctgt ccgactacga
     5761 tgtggaccat atcgtgcctc agagctttct gaaggacgac tccatcgaca acaaggtgct
     5821 gaccagaagc gacaagaacc ggggcaagag cgacaacgtg ccctccgaag aggtcgtgaa
     5881 gaagatgaag aactactggc ggcagctgct gaacgccaag ctgattaccc agagaaagtt
     5941 cgacaatctg accaaggccg agagaggcgg cctgagcgaa ctggataagg ccggcttcat
     6001 caagagacag ctggtggaaa cccggcagat cacaaagcac gtggcacaga tcctggactc
     6061 ccggatgaac actaagtacg acgagaatga caagctgatc cgggaagtga aagtgatcac
     6121 cctgaagtcc aagctggtgt ccgatttccg gaaggatttc cagttttaca aagtgcgcga
     6181 gatcaacaac taccaccacg cccacgacgc ctacctgaac gccgtcgtgg gaaccgccct
     6241 gatcaaaaag taccctaagc tggaaagcga gttcgtgtac ggcgactaca aggtgtacga
     6301 cgtgcggaag atgatcgcca agagcgagca ggaaatcggc aaggctaccg ccaagtactt
     6361 cttctacagc aacatcatga actttttcaa gaccgagatt accctggcca acggcgagat
     6421 ccggaagcgg cctctgatcg agacaaacgg cgaaaccggg gagatcgtgt gggataaggg
     6481 ccgggatttt gccaccgtgc ggaaagtgct gagcatgccc caagtgaata tcgtgaaaaa
     6541 gaccgaggtg cagacaggcg gcttcagcaa agagtctatc ctgcccaaga ggaacagcga
     6601 taagctgatc gccagaaaga aggactggga ccctaagaag tacggcggct tcgacagccc
     6661 caccgtggcc tattctgtgc tggtggtggc caaagtggaa aagggcaagt ccaagaaact
     6721 gaagagtgtg aaagagctgc tggggatcac catcatggaa agaagcagct tcgagaagaa
     6781 tcccatcgac tttctggaag ccaagggcta caaagaagtg aaaaaggacc tgatcatcaa
     6841 gctgcctaag tactccctgt tcgagctgga aaacggccgg aagagaatgc tggcctctgc
     6901 cggcgaactg cagaagggaa acgaactggc cctgccctcc aaatatgtga acttcctgta
     6961 cctggccagc cactatgaga agctgaaggg ctcccccgag gataatgagc agaaacagct
     7021 gtttgtggaa cagcacaagc actacctgga cgagatcatc gagcagatca gcgagttctc
     7081 caagagagtg atcctggccg acgctaatct ggacaaagtg ctgtccgcct acaacaagca
     7141 ccgggataag cccatcagag agcaggccga gaatatcatc cacctgttta ccctgaccaa
     7201 tctgggagcc cctgccgcct tcaagtactt tgacaccacc atcgaccgga agaggtacac
     7261 cagcaccaaa gaggtgctgg acgccaccct gatccaccag agcatcaccg gcctgtacga
     7321 gacacggatc gacctgtctc agctgggagg cgacaagcga cctgccgcca caaagaaggc
     7381 tggacaggct aagaagaaga aagattacaa agacgatgac gataagggct ctagaggatc
     7441 cgttaactaa cttaagctag caacggtttc cctctagcgg gatcaattcc gccccccccc
     7501 cctaacgtta ctggccgaag ccgcttggaa taaggccggt gtgcgtttgt ctatatgtta
     7561 ttttccacca tattgccgtc ttttggcaat gtgagggccc ggaaacctgg ccctgtcttc
     7621 ttgacgagca ttcctagggg tctttcccct ctcgccaaag gaatgcaagg tctgttgaat
     7681 gtcgtgaagg aagcagttcc tctggaagct tcttgaagac aaacaacgtc tgtagcgacc
     7741 ctttgcaggc agcggaaccc cccacctggc gacaggtgcc tctgcggcca aaagccacgt
     7801 gtataagata cacctgcaaa ggcggcacaa ccccagtgcc acgttgtgag ttggatagtt
     7861 gtggaaagag tcaaatggct ctcctcaagc gtattcaaca aggggctgaa ggatgcccag
     7921 aaggtacccc attgtatggg atctgatctg gggcctcggt gcacatgctt tacatgtgtt
     7981 tagtcgaggt taaaaaaacg tctaggcccc ccgaaccacg gggacgtggt tttcctttga
     8041 aaaacacgat aataccatgg ccaagttgac cagtgccgtt ccggtgctca ccgcgcgcga
     8101 cgtcgccgga gcggtcgagt tctggaccga ccggctcggg ttctcccggg acttcgtgga
     8161 ggacgacttc gccggtgtgg tccgggacga cgtgaccctg ttcatcagcg cggtccagga
     8221 ccaggtggtg ccggacaaca ccctggcctg ggtgtgggtg cgcggcctgg acgagctgta
     8281 cgccgagtgg tcggaggtcg tgtccacgaa cttccgggac gcctccgggc cggccatgac
     8341 cgagatcggc gagcagccgt gggggcggga gttcgccctg cgcgacccgg ccggcaactg
     8401 cgtgcacttc gtggccgagg agcaggactg aacgcgttaa gtcgacaatc aacctctgga
     8461 ttacaaaatt tgtgaaagat tgactggtat tcttaactat gttgctcctt ttacgctatg
     8521 tggatacgct gctttaatgc ctttgtatca tgctattgct tcccgtatgg ctttcatttt
     8581 ctcctccttg tataaatcct ggttgctgtc tctttatgag gagttgtggc ccgttgtcag
     8641 gcaacgtggc gtggtgtgca ctgtgtttgc tgacgcaacc cccactggtt ggggcattgc
     8701 caccacctgt cagctccttt ccgggacttt cgctttcccc ctccctattg ccacggcgga
     8761 actcatcgcc gcctgccttg cccgctgctg gacaggggct cggctgttgg gcactgacaa
     8821 ttccgtggtg ttgtcgggga aatcatcgtc ctttccttgg ctgctcgcct gtgttgccac
     8881 ctggattctg cgcgggacgt ccttctgcta cgtcccttcg gccctcaatc cagcggacct
     8941 tccttcccgc ggcctgctgc cggctctgcg gcctcttccg cgtcttcgcc ttcgccctca
     9001 gacgagtcgg atctcccttt gggccgcctc cccgcgtcga ctttaagacc aatgacttac
     9061 aaggcagctg tagatcttag ccacttttta aaagaaaagg ggggactgga agggctaatt
     9121 cactcccaac gaagacaaga tctgcttttt gcttgtactg ggtctctctg gttagaccag
     9181 atctgagcct gggagctctc tggctaacta gggaacccac tgcttaagcc tcaataaagc
     9241 ttgccttgag tgcttcaagt agtgtgtgcc cgtctgttgt gtgactctgg taactagaga
     9301 tccctcagac ccttttagtc agtgtggaaa atctctagca gggcccgttt aaacccgctg
     9361 atcagcctcg actgtgcctt ctagttgcca gccatctgtt gtttgcccct cccccgtgcc
     9421 ttccttgacc ctggaaggtg ccactcccac tgtcctttcc taataaaatg aggaaattgc
     9481 atcgcattgt ctgagtaggt gtcattctat tctggggggt ggggtggggc aggacagcaa
     9541 gggggaggat tgggaagaca atagcaggca tgctggggat gcggtgggct ctatggcttc
     9601 tgaggcggaa agaaccagct ggggctctag ggggtatccc cacgcgccct gtagcggcgc
     9661 attaagcgcg gcgggtgtgg tggttacgcg cagcgtgacc gctacacttg ccagcgccct
     9721 agcgcccgct cctttcgctt tcttcccttc ctttctcgcc acgttcgccg gctttccccg
     9781 tcaagctcta aatcgggggc tccctttagg gttccgattt agtgctttac ggcacctcga
     9841 ccccaaaaaa cttgattagg gtgatggttc acgtagtggg ccatcgccct gatagacggt
     9901 ttttcgccct ttgacgttgg agtccacgtt ctttaatagt ggactcttgt tccaaactgg
     9961 aacaacactc aaccctatct cggtctattc ttttgattta taagggattt tgccgatttc
    10021 ggcctattgg ttaaaaaatg agctgattta acaaaaattt aacgcgaatt aattctgtgg
    10081 aatgtgtgtc agttagggtg tggaaagtcc ccaggctccc cagcaggcag aagtatgcaa
    10141 agcatgcatc tcaattagtc agcaaccagg tgtggaaagt ccccaggctc cccagcaggc
    10201 agaagtatgc aaagcatgca tctcaattag tcagcaacca tagtcccgcc cctaactccg
    10261 cccatcccgc ccctaactcc gcccagttcc gcccattctc cgccccatgg ctgactaatt
    10321 ttttttattt atgcagaggc cgaggccgcc tctgcctctg agctattcca gaagtagtga
    10381 ggaggctttt ttggaggcct aggcttttgc aaaaagctcc cgggagcttg tatatccatt
    10441 ttcggatctg atcagcacgt gttgacaatt aatcatcggc atagtatatc ggcatagtat
    10501 aatacgacaa ggtgaggaac taaaccatgg ccaagttgac cagtgccgtt ccggtgctca
    10561 ccgcgcgcga cgtcgccgga gcggtcgagt tctggaccga ccggctcggg ttctcccggg
    10621 acttcgtgga ggacgacttc gccggtgtgg tccgggacga cgtgaccctg ttcatcagcg
    10681 cggtccagga ccaggtggtg ccggacaaca ccctggcctg ggtgtgggtg cgcggcctgg
    10741 acgagctgta cgccgagtgg tcggaggtcg tgtccacgaa cttccgggac gcctccgggc
    10801 cggccatgac cgagatcggc gagcagccgt gggggcggga gttcgccctg cgcgacccgg
    10861 ccggcaactg cgtgcacttc gtggccgagg agcaggactg acacgtgcta cgagatttcg
    10921 attccaccgc cgccttctat gaaaggttgg gcttcggaat cgttttccgg gacgccggct
    10981 ggatgatcct ccagcgcggg gatctcatgc tggagttctt cgcccacccc aacttgttta
    11041 ttgcagctta taatggttac aaataaagca atagcatcac aaatttcaca aataaagcat
    11101 ttttttcact gcattctagt tgtggtttgt ccaaactcat caatgtatct tatcatgtct
    11161 gtataccgtc gacctctagc tagagcttgg cgtaatcatg gtcatagctg tttcctgtgt
    11221 gaaattgtta tccgctcaca attccacaca acatacgagc cggaagcata aagtgtaaag
    11281 cctggggtgc ctaatgagtg agctaactca cattaattgc gttgcgctca ctgcccgctt
    11341 tccagtcggg aaacctgtcg tgccagctgc attaatgaat cggccaacgc gcggggagag
    11401 gcggtttgcg tattgggcgc tcttccgctt cctcgctcac tgactcgctg cgctcggtcg
    11461 ttcggctgcg gcgagcggta tcagctcact caaaggcggt aatacggtta tccacagaat
    11521 caggggataa cgcaggaaag aacatgtgag caaaaggcca gcaaaaggcc aggaaccgta
    11581 aaaaggccgc gttgctggcg tttttccata ggctccgccc ccctgacgag catcacaaaa
    11641 atcgacgctc aagtcagagg tggcgaaacc cgacaggact ataaagatac caggcgtttc
    11701 cccctggaag ctccctcgtg cgctctcctg ttccgaccct gccgcttacc ggatacctgt
    11761 ccgcctttct cccttcggga agcgtggcgc tttctcatag ctcacgctgt aggtatctca
    11821 gttcggtgta ggtcgttcgc tccaagctgg gctgtgtgca cgaacccccc gttcagcccg
    11881 accgctgcgc cttatccggt aactatcgtc ttgagtccaa cccggtaaga cacgacttat
    11941 cgccactggc agcagccact ggtaacagga ttagcagagc gaggtatgta ggcggtgcta
    12001 cagagttctt gaagtggtgg cctaactacg gctacactag aagaacagta tttggtatct
    12061 gcgctctgct gaagccagtt accttcggaa aaagagttgg tagctcttga tccggcaaac
    12121 aaaccaccgc tggtagcggt ggtttttttg tttgcaagca gcagattacg cgcagaaaaa
    12181 aaggatctca agaagatcct ttgatctttt ctacggggtc tgacgctcag tggaacgaaa
    12241 actcacgtta agggattttg gtcatgagat tatcaaaaag gatcttcacc tagatccttt
    12301 taaattaaaa atgaagtttt aaatcaatct aaagtatata tgagtaaact tggtctgaca
    12361 gttaccaatg cttaatcagt gaggcaccta tctcagcgat ctgtctattt cgttcatcca
    12421 tagttgcctg actccccgtc gtgtagataa ctacgatacg ggagggctta ccatctggcc
    12481 ccagtgctgc aatgataccg cgagacccac gctcaccggc tccagattta tcagcaataa
    12541 accagccagc cggaagggcc gagcgcagaa gtggtcctgc aactttatcc gcctccatcc
    12601 agtctattaa ttgttgccgg gaagctagag taagtagttc gccagttaat agtttgcgca
    12661 acgttgttgc cattgctaca ggcatcgtgg tgtcacgctc gtcgtttggt atggcttcat
    12721 tcagctccgg ttcccaacga tcaaggcgag ttacatgatc ccccatgttg tgcaaaaaag
    12781 cggttagctc cttcggtcct ccgatcgttg tcagaagtaa gttggccgca gtgttatcac
    12841 tcatggttat ggcagcactg cataattctc ttactgtcat gccatccgta agatgctttt
    12901 ctgtgactgg tgagtactca accaagtcat tctgagaata gtgtatgcgg cgaccgagtt
    12961 gctcttgccc ggcgtcaata cgggataata ccgcgccaca tagcagaact ttaaaagtgc
    13021 tcatcattgg aaaacgttct tcggggcgaa aactctcaag gatcttaccg ctgttgagat
    13081 ccagttcgat gtaacccact cgtgcaccca actgatcttc agcatctttt actttcacca
    13141 gcgtttctgg gtgagcaaaa acaggaaggc aaaatgccgc aaaaaaggga ataagggcga
    13201 cacggaaatg ttgaatactc atactcttcc tttttcaata ttattgaagc atttatcagg
    13261 gttattgtct catgagcgga tacatatttg aatgtattta gaaaaataaa caaatagggg
    13321 ttccgcgcac atttccccga aaagtgccac ctgac
//
