LOCUS       sequence-178234-       10183 bp    DNA     circular SYN 15-DEC-2025
DEFINITION  CAG-driven ubiquitous expression of catalytically inactive Cas9 
            fused to KRAB repressor domain. Contains 2A-EGFP reporter. For 
            targeted enhancer inactivation in chicken embryos..
ACCESSION   .
VERSION     .
KEYWORDS    .
SOURCE      synthetic DNA construct
  ORGANISM  synthetic DNA construct
REFERENCE   1  (bases 1 to 10183)
  AUTHORS   Williams RM, Senanayake U, Artibani M, Taylor G, Wells D, Ahmed AA, 
            Sauka-Spengler T
  TITLE     Genome and epigenome engineering CRISPR toolkit for in vivo 
            modulation of cis-regulatory interactions and gene expression in the
            chicken embryo.
  JOURNAL   Development. 2018 Feb 23;145(4). pii: dev.160333. doi: 
            10.1242/dev.160333.
  PUBMED    29386245
REFERENCE   2  (bases 1 to 10183)
  AUTHORS   .
  TITLE     Direct Submission
  JOURNAL   Exported Dec 15, 2025 from SnapGene Server 8.0.1
            https://www.snapgene.com
COMMENT     SGRef: number: 1; type: "Journal Article"; journalName: 
            "Development."; date: "2018-02-23"; volume: "145(4). pii: 
            dev.160333. doi"; pages: " 10.1242/dev.160333"
COMMENT     Sequence Label: pCAG dCas9-KRAB-2A-EGFP
FEATURES             Location/Qualifiers
     source          1..10183
                     /mol_type="other DNA"
                     /organism="synthetic DNA construct"
     enhancer        5..384
                     /label=CMV enhancer
                     /note="human cytomegalovirus immediate early enhancer"
     promoter        386..661
                     /label=chicken beta-actin promoter
     intron          662..1674
                     /label=chimeric intron
                     /note="chimera between introns from chicken beta-actin and 
                     rabbit beta-globin"
     primer_bind     1598..1620
                     /label=pCAGGS-5
                     /note="Chimeric intron in CAG promoter, forward primer"
     primer_bind     1682..1701
                     /label=pCAG-F
                     /note="Rabbit beta-globin intron, for pCAG plasmids, 
                     forward primer"
     regulatory      1734..1743
                     /label=Kozak sequence
                     /note="vertebrate consensus sequence for strong initiation 
                     of translation (Kozak, 1987)"
                     /regulatory_class="other"
     CDS             1743..1808
                     /codon_start=1
                     /product="three tandem FLAG(R) epitope tags, followed by an
                     enterokinase cleavage site"
                     /label=3xFLAG
                     /translation="DYKDHDGDYKDHDIDYKDDDDK"
     CDS             1815..1835
                     /codon_start=1
                     /product="nuclear localization signal of SV40 (simian virus
                     40) large T antigen"
                     /label=SV40 NLS
                     /translation="PKKKRKV"
     CDS             join(1845..1871,1872..1874,1875..4358,4359..4364,4365..4430,
                     4431..4433,4434..5948)
                     /codon_start=1
                     /product="catalytically dead mutant of the Cas9 
                     endonuclease from the Streptococcus pyogenes Type II 
                     CRISPR/Cas system"
                     /label=Cas9m4
                     /note="RNA-guided DNA-binding protein that lacks 
                     endonuclease activity due to the mutations D10A, D839A, 
                     H840A, and N863A (Mali et al., 2013)"
                     /translation="MDKKYSIGLAIGTNSVGWAVITDEYKVPSKKFKVLGNTDRHSIKK
                     NLIGALLFDSGETAEATRLKRTARRRYTRRKNRICYLQEIFSNEMAKVDDSFFHRLEES
                     FLVEEDKKHERHPIFGNIVDEVAYHEKYPTIYHLRKKLVDSTDKADLRLIYLALAHMIK
                     FRGHFLIEGDLNPDNSDVDKLFIQLVQTYNQLFEENPINASGVDAKAILSARLSKSRRL
                     ENLIAQLPGEKKNGLFGNLIALSLGLTPNFKSNFDLAEDAKLQLSKDTYDDDLDNLLAQ
                     IGDQYADLFLAAKNLSDAILLSDILRVNTEITKAPLSASMIKRYDEHHQDLTLLKALVR
                     QQLPEKYKEIFFDQSKNGYAGYIDGGASQEEFYKFIKPILEKMDGTEELLVKLNREDLL
                     RKQRTFDNGSIPHQIHLGELHAILRRQEDFYPFLKDNREKIEKILTFRIPYYVGPLARG
                     NSRFAWMTRKSEETITPWNFEEVVDKGASAQSFIERMTNFDKNLPNEKVLPKHSLLYEY
                     FTVYNELTKVKYVTEGMRKPAFLSGEQKKAIVDLLFKTNRKVTVKQLKEDYFKKIECFD
                     SVEISGVEDRFNASLGTYHDLLKIIKDKDFLDNEENEDILEDIVLTLTLFEDREMIEER
                     LKTYAHLFDDKVMKQLKRRRYTGWGRLSRKLINGIRDKQSGKTILDFLKSDGFANRNFM
                     QLIHDDSLTFKEDIQKAQVSGQGDSLHEHIANLAGSPAIKKGILQTVKVVDELVKVMGR
                     HKPENIVIEMARENQTTQKGQKNSRERMKRIEEGIKELGSQILKEHPVENTQLQNEKLY
                     LYYLQNGRDMYVDQELDINRLSDYDVDAIVPQSFLKDDSIDNKVLTRSDKNRGKSDNVP
                     SEEVVKKMKNYWRQLLNAKLITQRKFDNLTKAERGGLSELDKAGFIKRQLVETRQITKH
                     VAQILDSRMNTKYDENDKLIREVKVITLKSKLVSDFRKDFQFYKVREINNYHHAHDAYL
                     NAVVGTALIKKYPKLESEFVYGDYKVYDVRKMIAKSEQEIGKATAKYFFYSNIMNFFKT
                     EITLANGEIRKRPLIETNGETGEIVWDKGRDFATVRKVLSMPQVNIVKKTEVQTGGFSK
                     ESILPKRNSDKLIARKKDWDPKKYGGFDSPTVAYSVLVVAKVEKGKSKKLKSVKELLGI
                     TIMERSSFEKNPIDFLEAKGYKEVKKDLIIKLPKYSLFELENGRKRMLASAGELQKGNE
                     LALPSKYVNFLYLASHYEKLKGSPEDNEQKQLFVEQHKHYLDEIIEQISEFSKRVILAD
                     ANLDKVLSAYNKHRDKPIREQAENIIHLFTLTNLGAPAAFKYFDTTIDRKRYTSTKEVL
                     DATLIHQSITGLYETRIDLSQLGGD"
     CDS             join(1845..1871,1872..1874,1875..4361,4362..4364,
                     4365..5948)
                     /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             1845..5948
                     /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             5961..5981
                     /codon_start=1
                     /product="nuclear localization signal of SV40 (simian virus
                     40) large T antigen"
                     /label=SV40 NLS
                     /translation="PKKKRKV"
     CDS             6015..6209
                     /codon_start=1
                     /product="Kruppel-associated box (KRAB) transcriptional 
                     repression domain from the human zinc finger protein ZNF10 
                     (Margolin et al., 1994)"
                     /label=KRAB
                     /translation="RTLVTFKDVFVDFTREEWKLLDTAQQILYRNVMLENYKNLVSLGY
                     QLTKPDVILRLEKGEEPWLV"
     CDS             6276..6296
                     /codon_start=1
                     /product="nuclear localization signal of SV40 (simian virus
                     40) large T antigen"
                     /label=SV40 NLS
                     /translation="PKKKRKV"
     CDS             6303..6356
                     /codon_start=1
                     /product="2A peptide from Thosea asigna virus capsid 
                     protein"
                     /label=T2A
                     /note="Eukaryotic ribosomes fail to insert a peptide bond 
                     between the Gly and Pro residues, yielding separate 
                     polypeptides."
                     /translation="EGRGSLLTCGDVEENPGP"
     CDS             join(6363..6365,6366..7076)
                     /codon_start=1
                     /product="enhanced GFP"
                     /label=EGFP
                     /translation="VSKGEELFTGVVPILVELDGDVNGHKFSVSGEGEGDATYGKLTLK
                     FICTTGKLPVPWPTLVTTLTYGVQCFSRYPDHMKQHDFFKSAMPEGYVQERTIFFKDDG
                     NYKTRAEVKFEGDTLVNRIELKGIDFKEDGNILGHKLEYNYNSHNVYIMADKQKNGIKV
                     NFKIRHNIEDGSVQLADHYQQNTPIGDGPVLLPDNHYLSTQSALSKDPNEKRDHMVLLE
                     FVTAAGITLGMDELYK"
     CDS             join(6363..6365,6366..7076)
                     /codon_start=1
                     /product="the original enhanced GFP (Yang et al., 1996)"
                     /label=EGFP
                     /note="mammalian codon-optimized"
                     /translation="VSKGEELFTGVVPILVELDGDVNGHKFSVSGEGEGDATYGKLTLK
                     FICTTGKLPVPWPTLVTTLTYGVQCFSRYPDHMKQHDFFKSAMPEGYVQERTIFFKDDG
                     NYKTRAEVKFEGDTLVNRIELKGIDFKEDGNILGHKLEYNYNSHNVYIMADKQKNGIKV
                     NFKIRHNIEDGSVQLADHYQQNTPIGDGPVLLPDNHYLSTQSALSKDPNEKRDHMVLLE
                     FVTAAGITLGMDELYK"
     primer_bind     complement(6405..6426)
                     /label=EGFP-N
                     /note="EGFP, reverse primer"
     primer_bind     complement(6666..6685)
                     /label=EXFP-R
                     /note="For distinguishing EGFP variants, reverse primer"
     primer_bind     7013..7034
                     /label=EGFP-C
                     /note="EGFP, forward primer"
     primer_bind     complement(7175..7194)
                     /label=Bglob-pA-R
                     /note="Rabbit beta-globin polyA region, reverse primer"
     polyA_signal    7240..7295
                     /label=beta-globin poly(A) signal
                     /note="rabbit beta-globin polyadenylation signal (Gil and 
                     Proudfoot, 1987)"
     primer_bind     complement(7294..7313)
                     /label=rbglobpA-R
                     /note="Rabbit beta-globin polyA, reverse primer. Also 
                     called rb-glob-pA-term-R"
     primer_bind     complement(7686..7702)
                     /label=M13 rev
                     /note="common sequencing primer, one of multiple similar 
                     variants"
     primer_bind     complement(7686..7702)
                     /label=M13 Reverse
                     /note="In lacZ gene. Also called M13-rev"
     primer_bind     complement(7699..7721)
                     /label=M13/pUC Reverse
                     /note="In lacZ gene"
     protein_bind    7710..7726
                     /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(7734..7740,7741..7758,7759..7764))
                     /label=lac promoter
                     /note="promoter for the E. coli lac operon"
     protein_bind    7779..7800
                     /label=CAP binding site
                     /bound_moiety="E. coli catabolite activator protein"
                     /note="CAP binding activates transcription in the presence 
                     of cAMP."
     promoter        7859..8054
                     /label=SV40 promoter
                     /note="SV40 early promoter"
     rep_origin      7905..8040
                     /label=SV40 ori
                     /note="SV40 origin of replication"
     polyA_signal    8060..8194
                     /label=SV40 poly(A) signal
                     /note="SV40 polyadenylation signal"
     primer_bind     complement(8097..8116)
                     /label=SV40pA-R
                     /note="SV40 polyA, reverse primer"
     primer_bind     8151..8170
                     /label=EBV-rev
                     /note="SV40 polyA terminator, reverse primer"
     primer_bind     complement(8262..8279)
                     /label=L4440
                     /note="L4440 vector, forward primer"
     rep_origin      complement(8433..9021)
                     /direction=LEFT
                     /label=ori
                     /note="high-copy-number ColE1/pMB1/pBR322/pUC origin of 
                     replication"
     primer_bind     complement(8513..8532)
                     /label=pBR322ori-F
                     /note="pBR322 origin, forward primer"
     CDS             complement(join(9192..9983,9984..10052))
                     /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     9815..9834
                     /label=Amp-R
                     /note="Ampicillin resistance gene, reverse primer"
     promoter        complement(10053..10157)
                     /gene="bla"
                     /label=AmpR promoter
ORIGIN
        1 ggtcgacatt gattattgac tagttattaa tagtaatcaa ttacggggtc attagttcat
       61 agcccatata tggagttccg cgttacataa cttacggtaa atggcccgcc tggctgaccg
      121 cccaacgacc cccgcccatt gacgtcaata atgacgtatg ttcccatagt aacgccaata
      181 gggactttcc attgacgtca atgggtggag tatttacggt aaactgccca cttggcagta
      241 catcaagtgt atcatatgcc aagtacgccc cctattgacg tcaatgacgg taaatggccc
      301 gcctggcatt atgcccagta catgacctta tgggactttc ctacttggca gtacatctac
      361 gtattagtca tcgctattac catggtcgag gtgagcccca cgttctgctt cactctcccc
      421 atctcccccc cctccccacc cccaattttg tatttattta ttttttaatt attttgtgca
      481 gcgatggggg cggggggggg gggggcgcgc gccaggcggg gcggggcggg gcgaggggcg
      541 gggcggggcg aggcggagag gtgcggcggc agccaatcag agcggcgcgc tccgaaagtt
      601 tccttttatg gcgaggcggc ggcggcggcg gccctataaa aagcgaagcg cgcggcgggc
      661 gggagtcgct gcgttgcctt cgccccgtgc cccgctccgc gccgcctcgc gccgcccgcc
      721 ccggctctga ctgaccgcgt tactcccaca ggtgagcggg cgggacggcc cttctcctcc
      781 gggctgtaat tagcgcttgg tttaatgacg gctcgtttct tttctgtggc tgcgtgaaag
      841 ccttaaaggg ctccgggagg gccctttgtg cgggggggag cggctcgggg ggtgcgtgcg
      901 tgtgtgtgtg cgtggggagc gccgcgtgcg gcccgcgctg cccggcggct gtgagcgctg
      961 cgggcgcggc gcggggcttt gtgcgctccg cgtgtgcgcg aggggagcgc ggccgggggc
     1021 ggtgccccgc ggtgcggggg ggctgcgagg ggaacaaagg ctgcgtgcgg ggtgtgtgcg
     1081 tgggggggtg agcagggggt gtgggcgcgg cggtcgggct gtaacccccc cctgcacccc
     1141 cctccccgag ttgctgagca cggcccggct tcgggtgcgg ggctccgtac ggggcgtggc
     1201 gcggggctcg ccgtgccggg cggggggtgg cggcaggtgg gggtgccggg cggggcgggg
     1261 ccgcctcggg ccggggaggg ctcgggggag gggcgcggcg gcccccggag cgccggcggc
     1321 tgtcgaggcg cggcgagccg cagccattgc cttttatggt aatcgtgcga gagggcgcag
     1381 ggacttcctt tgtcccaaat ctgtgcggag ccgaaatctg ggaggcgccg ccgcaccccc
     1441 tctagcgggc gcggggcgaa gcggtgcggc gccggcagga aggaaatggg cggggagggc
     1501 cttcgtgcgt cgccgcgccg ccgtcccctt ctccctctcc agcctcgggg ctgtccgcgg
     1561 ggggacggct gccttcgggg gggacggggc agggcggggt tcggcttctg gcgtgtgacc
     1621 ggcggctcta gagcctctgc taaccatgtt catgccttct tctttttcct acagctcctg
     1681 ggcaacgtgc tggttattgt gctgtctcat cattttggca aagaattgct cgagccacca
     1741 tggactacaa agaccatgac ggtgattata aagatcatga catcgattac aaggatgacg
     1801 atgacaagat ggcccccaag aagaagagga aggtgggccg cggaatggac aagaagtact
     1861 ccattgggct cgccatcggc acaaacagcg tcggctgggc cgtcattacg gacgagtaca
     1921 aggtgccgag caaaaaattc aaagttctgg gcaataccga tcgccacagc ataaagaaga
     1981 acctcattgg cgccctcctg ttcgactccg gggaaaccgc cgaagccacg cggctcaaaa
     2041 gaacagcacg gcgcagatat acccgcagaa agaatcggat ctgctacctg caggagatct
     2101 ttagtaatga gatggctaag gtggatgact ctttcttcca taggctggag gagtcctttt
     2161 tggtggagga ggataaaaag cacgagcgcc acccaatctt tggcaatatc gtggacgagg
     2221 tggcgtacca tgaaaagtac ccaaccatat atcatctgag gaagaagctt gtagacagta
     2281 ctgataaggc tgacttgcgg ttgatctatc tcgcgctggc gcatatgatc aaatttcggg
     2341 gacacttcct catcgagggg gacctgaacc cagacaacag cgatgtcgac aaactcttta
     2401 tccaactggt tcagacttac aatcagcttt tcgaagagaa cccgatcaac gcatccggag
     2461 ttgacgccaa agcaatcctg agcgctaggc tgtccaaatc ccggcggctc gaaaacctca
     2521 tcgcacagct ccctggggag aagaagaacg gcctgtttgg taatcttatc gccctgtcac
     2581 tcgggctgac ccccaacttt aaatctaact tcgacctggc cgaagatgcc aagcttcaac
     2641 tgagcaaaga cacctacgat gatgatctcg acaatctgct ggcccagatc ggcgaccagt
     2701 acgcagacct ttttttggcg gcaaagaacc tgtcagacgc cattctgctg agtgatattc
     2761 tgcgagtgaa cacggagatc accaaagctc cgctgagcgc tagtatgatc aagcgctatg
     2821 atgagcacca ccaagacttg actttgctga aggcccttgt cagacagcaa ctgcctgaga
     2881 agtacaagga aattttcttc gatcagtcta aaaatggcta cgccggatac attgacggcg
     2941 gagcaagcca ggaggaattt tacaaattta ttaagcccat cttggaaaaa atggacggca
     3001 ccgaggagct gctggtaaag cttaacagag aagatctgtt gcgcaaacag cgcactttcg
     3061 acaatggaag catcccccac cagattcacc tgggcgaact gcacgctatc ctcaggcggc
     3121 aagaggattt ctaccccttt ttgaaagata acagggaaaa gattgagaaa atcctcacat
     3181 ttcggatacc ctactatgta ggccccctcg cccggggaaa ttccagattc gcgtggatga
     3241 ctcgcaaatc agaagagacc atcactccct ggaacttcga ggaagtcgtg gataaggggg
     3301 cctctgccca gtccttcatc gaaaggatga ctaactttga taaaaatctg cctaacgaaa
     3361 aggtgcttcc taaacactct ctgctgtacg agtacttcac agtttataac gagctcacca
     3421 aggtcaaata cgtcacagaa gggatgagaa agccagcatt cctgtctgga gagcagaaga
     3481 aagctatcgt ggacctcctc ttcaagacga accggaaagt taccgtgaaa cagctcaaag
     3541 aagactattt caaaaagatt gaatgtttcg actctgttga aatcagcgga gtggaggatc
     3601 gcttcaacgc atccctggga acgtatcacg atctcctgaa aatcattaaa gacaaggact
     3661 tcctggacaa tgaggagaac gaggacattc ttgaggacat tgtcctcacc cttacgttgt
     3721 ttgaagatag ggagatgatt gaagaacgct tgaaaactta cgctcatctc ttcgacgaca
     3781 aagtcatgaa acagctcaag aggcgccgat atacaggatg ggggcggctg tcaagaaaac
     3841 tgatcaatgg gatccgagac aagcagagtg gaaagacaat cctggatttt cttaagtccg
     3901 atggatttgc caaccggaac ttcatgcagt tgatccatga tgactctctc acctttaagg
     3961 aggacatcca gaaagcacaa gtttctggcc agggggacag tcttcacgag cacatcgcta
     4021 atcttgcagg tagcccagct atcaaaaagg gaatactgca gaccgttaag gtcgtggatg
     4081 aactcgtcaa agtaatggga aggcataagc ccgagaatat cgttatcgag atggcccgag
     4141 agaaccaaac tacccagaag ggacagaaga acagtaggga aaggatgaag aggattgaag
     4201 agggtataaa agaactgggg tcccaaatcc ttaaggaaca cccagttgaa aacacccagc
     4261 ttcagaatga gaagctctac ctgtactacc tgcagaacgg cagggacatg tacgtggatc
     4321 aggaactgga catcaatcgg ctctccgact acgacgtgga tgccatcgtg ccccagtctt
     4381 ttctcaaaga tgattctatt gataataaag tgttgacaag atccgataaa aatagaggga
     4441 agagtgataa cgtcccctca gaagaagttg tcaagaaaat gaaaaattat tggcggcagc
     4501 tgctgaacgc caaactgatc acacaacgga agttcgataa tctgactaag gctgaacgag
     4561 gtggcctgtc tgagttggat aaagccggct tcatcaaaag gcagcttgtt gagacacgcc
     4621 agatcaccaa gcacgtggcc caaattctcg attcacgcat gaacaccaag tacgatgaaa
     4681 atgacaaact gattcgagag gtgaaagtta ttactctgaa gtctaagctg gtctcagatt
     4741 tcagaaagga ctttcagttt tataaggtga gagagatcaa caattaccac catgcgcatg
     4801 atgcctacct gaatgcagtg gtaggcactg cacttatcaa aaaatatccc aagcttgaat
     4861 ctgaatttgt ttacggagac tataaagtgt acgatgttag gaaaatgatc gcaaagtctg
     4921 agcaggaaat aggcaaggcc accgctaagt acttctttta cagcaatatt atgaattttt
     4981 tcaagaccga gattacactg gccaatggag agattcggaa gcgaccactt atcgaaacaa
     5041 acggagaaac aggagaaatc gtgtgggaca agggtaggga tttcgcgaca gtccggaagg
     5101 tcctgtccat gccgcaggtg aacatcgtta aaaagaccga agtacagacc ggaggcttct
     5161 ccaaggaaag tatcctcccg aaaaggaaca gcgacaagct gatcgcacgc aaaaaagatt
     5221 gggaccccaa gaaatacggc ggattcgatt ctcctacagt cgcttacagt gtactggttg
     5281 tggccaaagt ggagaaaggg aagtctaaaa aactcaaaag cgtcaaggaa ctgctgggca
     5341 tcacaatcat ggagcgatca agcttcgaaa aaaaccccat cgactttctc gaggcgaaag
     5401 gatataaaga ggtcaaaaaa gacctcatca ttaagcttcc caagtactct ctctttgagc
     5461 ttgaaaacgg ccggaaacga atgctcgcta gtgcgggcga gctgcagaaa ggtaacgagc
     5521 tggcactgcc ctctaaatac gttaatttct tgtatctggc cagccactat gaaaagctca
     5581 aagggtctcc cgaagataat gagcagaagc agctgttcgt ggaacaacac aaacactacc
     5641 ttgatgagat catcgagcaa ataagcgaat tctccaaaag agtgatcctc gccgacgcta
     5701 acctcgataa ggtgctttct gcttacaata agcacaggga taagcccatc agggagcagg
     5761 cagaaaacat tatccacttg tttactctga ccaacttggg cgcgcctgca gccttcaagt
     5821 acttcgacac caccatagac agaaagcggt acacctctac aaaggaggtc ctggacgcca
     5881 cactgattca tcagtcaatt acggggctct atgaaacaag aatcgacctc tctcagctcg
     5941 gtggagacag cagggctgac cccaagaaga agaggaaggt ggctagcgat gctaagtcac
     6001 tgactgcctg gtcccggaca ctggtgacct tcaaggatgt gtttgtggac ttcaccaggg
     6061 aggagtggaa gctgctggac actgctcagc agatcctgta cagaaatgtg atgctggaga
     6121 actataagaa cctggtttcc ttgggttatc agcttactaa gccagatgtg atcctccggt
     6181 tggagaaggg agaagagccc tggctggtgg agagagaaat tcaccaagag acccatcctg
     6241 attcagagac tgcatttgaa atcaaatcat cagttccgaa aaagaaacgc aaagttgcta
     6301 gcgagggcag aggaagtctt ctaacatgcg gtgacgtgga ggagaatccc ggccctggta
     6361 ccgtgagcaa gggcgaggag ctgttcaccg gggtggtgcc catcctggtc gagctggacg
     6421 gcgacgtaaa cggccacaag ttcagcgtgt ccggcgaggg cgagggcgat gccacctacg
     6481 gcaagctgac cctgaagttc atctgcacca ccggcaagct gcccgtgccc tggcccaccc
     6541 tcgtgaccac cctgacctac ggcgtgcagt gcttcagccg ctaccccgac cacatgaagc
     6601 agcacgactt cttcaagtcc gccatgcccg aaggctacgt ccaggagcgc accatcttct
     6661 tcaaggacga cggcaactac aagacccgcg ccgaggtgaa gttcgagggc gacaccctgg
     6721 tgaaccgcat cgagctgaag ggcatcgact tcaaggagga cggcaacatc ctggggcaca
     6781 agctggagta caactacaac agccacaacg tctatatcat ggccgacaag cagaagaacg
     6841 gcatcaaggt gaacttcaag atccgccaca acatcgagga cggcagcgtg cagctcgccg
     6901 accactacca gcagaacacc cccatcggcg acggccccgt gctgctgccc gacaaccact
     6961 acctgagcac ccagtccgcc ctgagcaaag accccaacga gaagcgcgat cacatggtcc
     7021 tgctggagtt cgtgaccgcc gccgggatca ctctcggcat ggacgagctg tacaagaccg
     7081 gttgatggcc gcaattcact cctcaggtgc aggctgccta tcagaaggtg gtggctggtg
     7141 tggccaatgc cctggctcac aaataccact gagatctttt tccctctgcc aaaaattatg
     7201 gggacatcat gaagcccctt gagcatctga cttctggcta ataaaggaaa tttattttca
     7261 ttgcaatagt gtgttggaat tttttgtgtc tctcactcgg aaggacatat gggagggcaa
     7321 atcatttaaa acatcagaat gagtatttgg tttagagttt ggcaacatat gcccatatgc
     7381 tggctgccat gaacaaaggt tggctataaa gaggtcatca gtatatgaaa cagccccctg
     7441 ctgtccattc cttattccat agaaaagcct tgacttgagg ttagattttt tttatatttt
     7501 gttttgtgtt atttttttct ttaacatccc taaaattttc cttacatgtt ttactagcca
     7561 gatttttcct cctctcctga ctactcccag tcatagctgt ccctcttctc ttatggagat
     7621 ccctcgacct gcaccgtcga ccagctggtc gacggtgcac cgtcgaccag cttggcgtaa
     7681 tcatggtcat agctgtttcc tgtgtgaaat tgttatccgc tcacaattcc acacaacata
     7741 cgagccggaa gcataaagtg taaagcctgg ggtgcctaat gagtgagcta actcacatta
     7801 attgcgttgc gctcactgcc cgctttccag tcgggaaacc tgtcgtgcca gcggatccgc
     7861 atctcaatta gtcagcaacc atagtcccgc ccctaactcc gcccatcccg cccctaactc
     7921 cgcccagttc cgcccattct ccgccccatg gctgactaat tttttttatt tatgcagagg
     7981 ccgaggccgc ctcggcctct gagctattcc agaagtagtg aggaggcttt tttggaggcc
     8041 taggcttttg caaaaagcta acttgtttat tgcagcttat aatggttaca aataaagcaa
     8101 tagcatcaca aatttcacaa ataaagcatt tttttcactg cattctagtt gtggtttgtc
     8161 caaactcatc aatgtatctt atcatgtctg gatccgctgc attaatgaat cggccaacgc
     8221 gcggggagag gcggtttgcg tattgggcgc tcttccgctt cctcgctcac tgactcgctg
     8281 cgctcggtcg ttcggctgcg gcgagcggta tcagctcact caaaggcggt aatacggtta
     8341 tccacagaat caggggataa cgcaggaaag aacatgtgag caaaaggcca gcaaaaggcc
     8401 aggaaccgta aaaaggccgc gttgctggcg tttttccata ggctccgccc ccctgacgag
     8461 catcacaaaa atcgacgctc aagtcagagg tggcgaaacc cgacaggact ataaagatac
     8521 caggcgtttc cccctggaag ctccctcgtg cgctctcctg ttccgaccct gccgcttacc
     8581 ggatacctgt ccgcctttct cccttcggga agcgtggcgc tttctcaatg ctcacgctgt
     8641 aggtatctca gttcggtgta ggtcgttcgc tccaagctgg gctgtgtgca cgaacccccc
     8701 gttcagcccg accgctgcgc cttatccggt aactatcgtc ttgagtccaa cccggtaaga
     8761 cacgacttat cgccactggc agcagccact ggtaacagga ttagcagagc gaggtatgta
     8821 ggcggtgcta cagagttctt gaagtggtgg cctaactacg gctacactag aaggacagta
     8881 tttggtatct gcgctctgct gaagccagtt accttcggaa aaagagttgg tagctcttga
     8941 tccggcaaac aaaccaccgc tggtagcggt ggtttttttg tttgcaagca gcagattacg
     9001 cgcagaaaaa aaggatctca agaagatcct ttgatctttt ctacggggtc tgacgctcag
     9061 tggaacgaaa actcacgtta agggattttg gtcatgagat tatcaaaaag gatcttcacc
     9121 tagatccttt taaattaaaa atgaagtttt aaatcaatct aaagtatata tgagtaaact
     9181 tggtctgaca gttaccaatg cttaatcagt gaggcaccta tctcagcgat ctgtctattt
     9241 cgttcatcca tagttgcctg actccccgtc gtgtagataa ctacgatacg ggagggctta
     9301 ccatctggcc ccagtgctgc aatgataccg cgagacccac gctcaccggc tccagattta
     9361 tcagcaataa accagccagc cggaagggcc gagcgcagaa gtggtcctgc aactttatcc
     9421 gcctccatcc agtctattaa ttgttgccgg gaagctagag taagtagttc gccagttaat
     9481 agtttgcgca acgttgttgc cattgctaca ggcatcgtgg tgtcacgctc gtcgtttggt
     9541 atggcttcat tcagctccgg ttcccaacga tcaaggcgag ttacatgatc ccccatgttg
     9601 tgcaaaaaag cggttagctc cttcggtcct ccgatcgttg tcagaagtaa gttggccgca
     9661 gtgttatcac tcatggttat ggcagcactg cataattctc ttactgtcat gccatccgta
     9721 agatgctttt ctgtgactgg tgagtactca accaagtcat tctgagaata gtgtatgcgg
     9781 cgaccgagtt gctcttgccc ggcgtcaata cgggataata ccgcgccaca tagcagaact
     9841 ttaaaagtgc tcatcattgg aaaacgttct tcggggcgaa aactctcaag gatcttaccg
     9901 ctgttgagat ccagttcgat gtaacccact cgtgcaccca actgatcttc agcatctttt
     9961 actttcacca gcgtttctgg gtgagcaaaa acaggaaggc aaaatgccgc aaaaaaggga
    10021 ataagggcga cacggaaatg ttgaatactc atactcttcc tttttcaata ttattgaagc
    10081 atttatcagg gttattgtct catgagcgga tacatatttg aatgtattta gaaaaataaa
    10141 caaatagggg ttccgcgcac atttccccga aaagtgccac ctg
//
