LOCUS       sequence-490395-       13648 bp    DNA     circular SYN 15-DEC-2025
DEFINITION  Cas9 protein with GFP.
ACCESSION   .
VERSION     .
KEYWORDS    .
SOURCE      synthetic DNA construct
  ORGANISM  synthetic DNA construct
REFERENCE   1  (bases 1 to 13648)
  AUTHORS   Pulido-Quetglas C, Aparicio-Prat E, Arnan C, Polidori T, Hermoso T, 
            Palumbo E, Ponomarenko J, Guigo R, Johnson R
  TITLE     Scalable Design of Paired CRISPR Guide RNAs for Genomic Deletion.
  JOURNAL   PLoS Comput Biol. 2017 Mar 2;13(3):e1005341. doi: 
            10.1371/journal.pcbi.1005341. eCollection 2017 Mar.
  PUBMED    28253259
REFERENCE   2  (bases 1 to 13648)
  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: "PLoS Comput
            Biol."; date: "2017-03-2"; volume: "13(3):e1005341. doi"; pages: " 
            10.1371/journal.pcbi.1005341. eCollection 2017 Mar"
COMMENT     Sequence Label: pLentiCas9-T2A-GFP
FEATURES             Location/Qualifiers
     source          1..13648
                     /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        2650..2861
                     /label=EF-1-alpha core promoter
                     /note="core promoter for human elongation factor 
                     EF-1-alpha"
     regulatory      2880..2889
                     /label=Kozak sequence
                     /note="vertebrate consensus sequence for strong initiation 
                     of translation (Kozak, 1987)"
                     /regulatory_class="other"
     CDS             2886..6989
                     /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             2886..6989
                     /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             6990..7037
                     /codon_start=1
                     /product="bipartite nuclear localization signal from 
                     nucleoplasmin"
                     /label=nucleoplasmin NLS
                     /translation="KRPAATKKAGQAKKKK"
     CDS             7038..7061
                     /codon_start=1
                     /product="FLAG(R) epitope tag, followed by an enterokinase 
                     cleavage site"
                     /label=FLAG
                     /translation="DYKDDDDK"
     CDS             7071..7124
                     /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"
     regulatory      7131..7140
                     /label=Kozak sequence
                     /note="vertebrate consensus sequence for strong initiation 
                     of translation (Kozak, 1987)"
                     /regulatory_class="other"
     CDS             join(7137..7139,7140..7142,7143..7853)
                     /codon_start=1
                     /product="the original enhanced GFP (Yang et al., 1996)"
                     /label=EGFP
                     /note="mammalian codon-optimized"
                     /translation="MVSKGEELFTGVVPILVELDGDVNGHKLSVSGEGEGDATYGKLTL
                     KFICTTGKLPVPWPTLVTTLTYGVQCFSRYPDHMKQHDFFKSAMPEGYVQERTIFFKDD
                     GNYKTRAEVKFEGDTLVNRIELKGIDFKEDGNILGHKLEYNYNSHNVYIMADKQKNGIK
                     VNFKIRHNIEDGSVQLADHYQQNTPIGDGPVLLPDNHYLSTQSALSKDPNEKRDHMVLL
                     EFVTAAGITLGMDELYK"
     primer_bind     complement(7443..7462)
                     /label=EXFP-R
                     /note="For distinguishing EGFP variants, reverse primer"
     primer_bind     7790..7811
                     /label=EGFP-C
                     /note="EGFP, forward primer"
     CDS             7854..7871
                     /codon_start=1
                     /product="6xHis affinity tag"
                     /label=6xHis
                     /translation="HHHHHH"
     CDS             7881..7937
                     /codon_start=1
                     /product="2A peptide from porcine teschovirus-1 
                     polyprotein"
                     /label=P2A
                     /note="Eukaryotic ribosomes fail to insert a peptide bond 
                     between the Gly and Pro residues, yielding separate 
                     polypeptides."
                     /translation="ATNFSLLKQAGDVEENPGP"
     CDS             7938..8336
                     /codon_start=1
                     /gene="Aspergillus terreus BSD"
                     /product="blasticidin S deaminase"
                     /label=BSD
                     /note="confers resistance to blasticidin"
                     /translation="MAKPLSQEESTLIERATATINSIPISEDYSVASAALSSDGRIFTG
                     VNVYHFTGGPCAELVVLGTAAAAAAGNLTCIVAIGNENRGILSPCGRCRQVLLDLHPGI
                     KAIVKDSDGQPTAVGIRELLPSGYVWEG"
     misc_feature    8361..8949
                     /label=WPRE
                     /note="woodchuck hepatitis virus posttranscriptional 
                     regulatory element"
     primer_bind     complement(8414..8434)
                     /label=WPRE-R
                     /note="WPRE, reverse primer"
     CDS             complement(8832..8843)
                     /codon_start=1
                     /product="Factor Xa recognition and cleavage site"
                     /label=Factor Xa site
                     /translation="IEGR"
     primer_bind     complement(8952..8968)
                     /label=KS primer
                     /note="common sequencing primer, one of multiple similar 
                     variants"
     primer_bind     complement(8953..8969)
                     /label=pBluescriptKS
                     /note="For pBluescript vector"
     LTR             9474..9654
                     /label=5' LTR (truncated)
                     /note="truncated 5' long terminal repeat (LTR) from HIV-1"
     primer_bind     complement(9680..9697)
                     /label=BGH-rev
                     /note="Bovine growth hormone terminator, reverse primer. 
                     Also called BGH reverse"
     polyA_signal    9686..9910
                     /label=bGH poly(A) signal
                     /note="bovine growth hormone polyadenylation signal"
     rep_origin      9956..10384
                     /direction=RIGHT
                     /label=f1 ori
                     /note="f1 bacteriophage origin of replication; arrow 
                     indicates direction of (+) strand synthesis"
     primer_bind     complement(10043..10062)
                     /label=F1ori-R
                     /note="F1 origin, reverse primer"
     primer_bind     10253..10274
                     /label=F1ori-F
                     /note="F1 origin, forward primer"
     primer_bind     complement(10393..10413)
                     /label=pBABE 3'
                     /note="SV40 enhancer, reverse primer for pBABE vectors"
     rep_origin      10558..10692
                     /label=SV40 ori
                     /note="SV40 origin of replication"
     primer_bind     10619..10638
                     /label=SV40pro-F
                     /note="SV40 promoter/origin, forward primer"
     promoter        10754..10801
                     /label=EM7 promoter
                     /note="synthetic bacterial promoter "
     CDS             10820..11194
                     /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    11324..11445
                     /label=SV40 poly(A) signal
                     /note="SV40 polyadenylation signal"
     primer_bind     complement(11361..11380)
                     /label=SV40pA-R
                     /note="SV40 polyA, reverse primer"
     primer_bind     11415..11434
                     /label=EBV-rev
                     /note="SV40 polyA terminator, reverse primer"
     primer_bind     complement(11494..11510)
                     /label=M13 rev
                     /note="common sequencing primer, one of multiple similar 
                     variants"
     primer_bind     complement(11494..11510)
                     /label=M13 Reverse
                     /note="In lacZ gene. Also called M13-rev"
     primer_bind     complement(11507..11529)
                     /label=M13/pUC Reverse
                     /note="In lacZ gene"
     protein_bind    11518..11534
                     /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(11542..11548,11549..11566,11567..11572))
                     /label=lac promoter
                     /note="promoter for the E. coli lac operon"
     protein_bind    11587..11608
                     /label=CAP binding site
                     /bound_moiety="E. coli catabolite activator protein"
                     /note="CAP binding activates transcription in the presence 
                     of cAMP."
     primer_bind     complement(11725..11742)
                     /label=L4440
                     /note="L4440 vector, forward primer"
     rep_origin      complement(11896..12484)
                     /direction=LEFT
                     /label=ori
                     /note="high-copy-number ColE1/pMB1/pBR322/pUC origin of 
                     replication"
     primer_bind     complement(11976..11995)
                     /label=pBR322ori-F
                     /note="pBR322 origin, forward primer"
     CDS             complement(join(12655..13446,13447..13515))
                     /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     13278..13297
                     /label=Amp-R
                     /note="Ampicillin resistance gene, reverse primer"
     promoter        complement(13516..13620)
                     /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 gctagctagg tcttgaaagg agtgggaatt ggctccggtg
     2641 cccgtcagtg ggcagagcgc acatcgccca cagtccccga gaagttgggg ggaggggtcg
     2701 gcaattgatc cggtgcctag agaaggtggc gcggggtaaa ctgggaaagt gatgtcgtgt
     2761 actggctccg cctttttccc gagggtgggg gagaaccgta tataagtgca gtagtcgccg
     2821 tgaacgttct ttttcgcaac gggtttgccg ccagaacaca ggaccggttc tagagcgctg
     2881 ccaccatgga caagaagtac agcatcggcc tggacatcgg caccaactct gtgggctggg
     2941 ccgtgatcac cgacgagtac aaggtgccca gcaagaaatt caaggtgctg ggcaacaccg
     3001 accggcacag catcaagaag aacctgatcg gagccctgct gttcgacagc ggcgaaacag
     3061 ccgaggccac ccggctgaag agaaccgcca gaagaagata caccagacgg aagaaccgga
     3121 tctgctatct gcaagagatc ttcagcaacg agatggccaa ggtggacgac agcttcttcc
     3181 acagactgga agagtccttc ctggtggaag aggataagaa gcacgagcgg caccccatct
     3241 tcggcaacat cgtggacgag gtggcctacc acgagaagta ccccaccatc taccacctga
     3301 gaaagaaact ggtggacagc accgacaagg ccgacctgcg gctgatctat ctggccctgg
     3361 cccacatgat caagttccgg ggccacttcc tgatcgaggg cgacctgaac cccgacaaca
     3421 gcgacgtgga caagctgttc atccagctgg tgcagaccta caaccagctg ttcgaggaaa
     3481 accccatcaa cgccagcggc gtggacgcca aggccatcct gtctgccaga ctgagcaaga
     3541 gcagacggct ggaaaatctg atcgcccagc tgcccggcga gaagaagaat ggcctgttcg
     3601 gaaacctgat tgccctgagc ctgggcctga cccccaactt caagagcaac ttcgacctgg
     3661 ccgaggatgc caaactgcag ctgagcaagg acacctacga cgacgacctg gacaacctgc
     3721 tggcccagat cggcgaccag tacgccgacc tgtttctggc cgccaagaac ctgtccgacg
     3781 ccatcctgct gagcgacatc ctgagagtga acaccgagat caccaaggcc cccctgagcg
     3841 cctctatgat caagagatac gacgagcacc accaggacct gaccctgctg aaagctctcg
     3901 tgcggcagca gctgcctgag aagtacaaag agattttctt cgaccagagc aagaacggct
     3961 acgccggcta cattgacggc ggagccagcc aggaagagtt ctacaagttc atcaagccca
     4021 tcctggaaaa gatggacggc accgaggaac tgctcgtgaa gctgaacaga gaggacctgc
     4081 tgcggaagca gcggaccttc gacaacggca gcatccccca ccagatccac ctgggagagc
     4141 tgcacgccat tctgcggcgg caggaagatt tttacccatt cctgaaggac aaccgggaaa
     4201 agatcgagaa gatcctgacc ttccgcatcc cctactacgt gggccctctg gccaggggaa
     4261 acagcagatt cgcctggatg accagaaaga gcgaggaaac catcaccccc tggaacttcg
     4321 aggaagtggt ggacaagggc gcttccgccc agagcttcat cgagcggatg accaacttcg
     4381 ataagaacct gcccaacgag aaggtgctgc ccaagcacag cctgctgtac gagtacttca
     4441 ccgtgtataa cgagctgacc aaagtgaaat acgtgaccga gggaatgaga aagcccgcct
     4501 tcctgagcgg cgagcagaaa aaggccatcg tggacctgct gttcaagacc aaccggaaag
     4561 tgaccgtgaa gcagctgaaa gaggactact tcaagaaaat cgagtgcttc gactccgtgg
     4621 aaatctccgg cgtggaagat cggttcaacg cctccctggg cacataccac gatctgctga
     4681 aaattatcaa ggacaaggac ttcctggaca atgaggaaaa cgaggacatt ctggaagata
     4741 tcgtgctgac cctgacactg tttgaggaca gagagatgat cgaggaacgg ctgaaaacct
     4801 atgcccacct gttcgacgac aaagtgatga agcagctgaa gcggcggaga tacaccggct
     4861 ggggcaggct gagccggaag ctgatcaacg gcatccggga caagcagtcc ggcaagacaa
     4921 tcctggattt cctgaagtcc gacggcttcg ccaacagaaa cttcatgcag ctgatccacg
     4981 acgacagcct gacctttaaa gaggacatcc agaaagccca ggtgtccggc cagggcgata
     5041 gcctgcacga gcacattgcc aatctggccg gcagccccgc cattaagaag ggcatcctgc
     5101 agacagtgaa ggtggtggac gagctcgtga aagtgatggg ccggcacaag cccgagaaca
     5161 tcgtgatcga aatggccaga gagaaccaga ccacccagaa gggacagaag aacagccgcg
     5221 agagaatgaa gcggatcgaa gagggcatca aagagctggg cagccagatc ctgaaagaac
     5281 accccgtgga aaacacccag ctgcagaacg agaagctgta cctgtactac ctgcagaatg
     5341 ggcgggatat gtacgtggac caggaactgg acatcaaccg gctgtccgac tacgatgtgg
     5401 accatatcgt gcctcagagc tttctgaagg acgactccat cgacaacaag gtgctgacca
     5461 gaagcgacaa gaaccggggc aagagcgaca acgtgccctc cgaagaggtc gtgaagaaga
     5521 tgaagaacta ctggcggcag ctgctgaacg ccaagctgat tacccagaga aagttcgaca
     5581 atctgaccaa ggccgagaga ggcggcctga gcgaactgga taaggccggc ttcatcaaga
     5641 gacagctggt ggaaacccgg cagatcacaa agcacgtggc acagatcctg gactcccgga
     5701 tgaacactaa gtacgacgag aatgacaagc tgatccggga agtgaaagtg atcaccctga
     5761 agtccaagct ggtgtccgat ttccggaagg atttccagtt ttacaaagtg cgcgagatca
     5821 acaactacca ccacgcccac gacgcctacc tgaacgccgt cgtgggaacc gccctgatca
     5881 aaaagtaccc taagctggaa agcgagttcg tgtacggcga ctacaaggtg tacgacgtgc
     5941 ggaagatgat cgccaagagc gagcaggaaa tcggcaaggc taccgccaag tacttcttct
     6001 acagcaacat catgaacttt ttcaagaccg agattaccct ggccaacggc gagatccgga
     6061 agcggcctct gatcgagaca aacggcgaaa ccggggagat cgtgtgggat aagggccggg
     6121 attttgccac cgtgcggaaa gtgctgagca tgccccaagt gaatatcgtg aaaaagaccg
     6181 aggtgcagac aggcggcttc agcaaagagt ctatcctgcc caagaggaac agcgataagc
     6241 tgatcgccag aaagaaggac tgggacccta agaagtacgg cggcttcgac agccccaccg
     6301 tggcctattc tgtgctggtg gtggccaaag tggaaaaggg caagtccaag aaactgaaga
     6361 gtgtgaaaga gctactgggg atcaccatca tggaaagaag cagcttcgag aagaatccca
     6421 tcgactttct ggaagccaag ggctacaaag aagtgaaaaa ggacctgatc atcaagctgc
     6481 ctaagtactc cctgttcgag ctggaaaacg gccggaagag aatgctggcc tctgccggcg
     6541 aactgcagaa gggaaacgaa ctggccctgc cctccaaata tgtgaacttc ctgtacctgg
     6601 ccagccacta tgagaagctg aagggctccc ccgaggataa tgagcagaaa cagctgtttg
     6661 tggaacagca caagcactac ctggacgaga tcatcgagca gatcagcgag ttctccaaga
     6721 gagtgatcct ggccgacgct aatctggaca aagtgctgtc cgcctacaac aagcaccggg
     6781 ataagcccat cagagagcag gccgagaata tcatccacct gtttaccctg accaatctgg
     6841 gagcccctgc cgccttcaag tactttgaca ccaccatcga ccggaagagg tacaccagca
     6901 ccaaagaggt gctggacgcc accctgatcc accagagcat caccggcctg tacgagacac
     6961 ggatcgacct gtctcagctg ggaggcgaca agcgacctgc cgccacaaag aaggctggac
     7021 aggctaagaa gaagaaagat tacaaagacg atgacgataa gggatccgga gagggcagag
     7081 gaagtctgct aacatgcggt gacgtcgagg agaatcctgg cccaccggtc gccaccatgg
     7141 tgagcaaggg cgaggagctg ttcaccgggg tggtgcccat cctggtcgag ctggacggcg
     7201 acgtaaacgg ccacaagctc agcgtgtccg gcgagggcga gggcgatgcc acctacggca
     7261 agctgaccct gaagttcatc tgcaccaccg gcaagctgcc cgtgccctgg cccaccctcg
     7321 tgaccaccct gacctacggc gtgcagtgct tcagccgcta ccccgaccac atgaagcagc
     7381 acgacttctt caagtccgcc atgcccgaag gctacgtcca ggagcgcacc atcttcttca
     7441 aggacgacgg caactacaag acccgcgccg aggtgaagtt cgagggcgac accctggtga
     7501 accgcatcga gctgaagggc atcgacttca aggaggacgg caacatcctg gggcacaagc
     7561 tggagtacaa ctacaacagc cacaacgtct atatcatggc cgacaagcag aagaacggca
     7621 tcaaggtgaa cttcaagatc cgccacaaca tcgaggacgg cagcgtgcag ctcgccgacc
     7681 actaccagca gaacaccccc atcggcgacg gccccgtgct gctgcccgac aaccactacc
     7741 tgagcaccca gtccgccctg agcaaagacc ccaacgagaa gcgcgatcac atggtcctgc
     7801 tggagttcgt gaccgccgcc gggatcactc tcggcatgga cgagctgtac aagcatcatc
     7861 accatcacca cggatccggc gcaacaaact tctctctgct gaaacaagcc ggagatgtcg
     7921 aagagaatcc tggaccgatg gccaagcctt tgtctcaaga agaatccacc ctcattgaaa
     7981 gagcaacggc tacaatcaac agcatcccca tctctgaaga ctacagcgtc gccagcgcag
     8041 ctctctctag cgacggccgc atcttcactg gtgtcaatgt atatcatttt actgggggac
     8101 cttgtgcaga actcgtggtg ctgggcactg ctgctgctgc ggcagctggc aacctgactt
     8161 gtatcgtcgc gatcggaaat gagaacaggg gcatcttgag cccctgcgga cggtgccgac
     8221 aggtgcttct cgatctgcat cctgggatca aagccatagt gaaggacagt gatggacagc
     8281 cgacggcagt tgggattcgt gaattgctgc cctctggtta tgtgtgggag ggctaagaat
     8341 tcgatatcaa gcttatcggt aatcaacctc tggattacaa aatttgtgaa agattgactg
     8401 gtattcttaa ctatgttgct ccttttacgc tatgtggata cgctgcttta atgcctttgt
     8461 atcatgctat tgcttcccgt atggctttca ttttctcctc cttgtataaa tcctggttgc
     8521 tgtctcttta tgaggagttg tggcccgttg tcaggcaacg tggcgtggtg tgcactgtgt
     8581 ttgctgacgc aacccccact ggttggggca ttgccaccac ctgtcagctc ctttccggga
     8641 ctttcgcttt ccccctccct attgccacgg cggaactcat cgccgcctgc cttgcccgct
     8701 gctggacagg ggctcggctg ttgggcactg acaattccgt ggtgttgtcg gggaaatcat
     8761 cgtcctttcc ttggctgctc gcctgtgttg ccacctggat tctgcgcggg acgtccttct
     8821 gctacgtccc ttcggccctc aatccagcgg accttccttc ccgcggcctg ctgccggctc
     8881 tgcggcctct tccgcgtctt cgccttcgcc ctcagacgag tcggatctcc ctttgggccg
     8941 cctccccgca tcgataccgt cgacctcgag acctagaaaa acatggagca atcacaagta
     9001 gcaatacagc agctaccaat gctgattgtg cctggctaga agcacaagag gaggaggagg
     9061 tgggttttcc agtcacacct caggtacctt taagaccaat gacttacaag gcagctgtag
     9121 atcttagcca ctttttaaaa gaaaaggggg gactggaagg gctaattcac tcccaacgaa
     9181 gacaagatat ccttgatctg tggatctacc acacacaagg ctacttccct gattggcaga
     9241 actacacacc agggccaggg atcagatatc cactgacctt tggatggtgc tacaagctag
     9301 taccagttga gcaagagaag gtagaagaag ccaatgaagg agagaacacc cgcttgttac
     9361 accctgtgag cctgcatggg atggatgacc cggagagaga agtattagag tggaggtttg
     9421 acagccgcct agcatttcat cacatggccc gagagctgca tccggactgt actgggtctc
     9481 tctggttaga ccagatctga gcctgggagc tctctggcta actagggaac ccactgctta
     9541 agcctcaata aagcttgcct tgagtgcttc aagtagtgtg tgcccgtctg ttgtgtgact
     9601 ctggtaacta gagatccctc agaccctttt agtcagtgtg gaaaatctct agcagggccc
     9661 gtttaaaccc gctgatcagc ctcgactgtg ccttctagtt gccagccatc tgttgtttgc
     9721 ccctcccccg tgccttcctt gaccctggaa ggtgccactc ccactgtcct ttcctaataa
     9781 aatgaggaaa ttgcatcgca ttgtctgagt aggtgtcatt ctattctggg gggtggggtg
     9841 gggcaggaca gcaaggggga ggattgggaa gacaatagca ggcatgctgg ggatgcggtg
     9901 ggctctatgg cttctgaggc ggaaagaacc agctggggct ctagggggta tccccacgcg
     9961 ccctgtagcg gcgcattaag cgcggcgggt gtggtggtta cgcgcagcgt gaccgctaca
    10021 cttgccagcg ccctagcgcc cgctcctttc gctttcttcc cttcctttct cgccacgttc
    10081 gccggctttc cccgtcaagc tctaaatcgg gggctccctt tagggttccg atttagtgct
    10141 ttacggcacc tcgaccccaa aaaacttgat tagggtgatg gttcacgtag tgggccatcg
    10201 ccctgataga cggtttttcg ccctttgacg ttggagtcca cgttctttaa tagtggactc
    10261 ttgttccaaa ctggaacaac actcaaccct atctcggtct attcttttga tttataaggg
    10321 attttgccga tttcggccta ttggttaaaa aatgagctga tttaacaaaa atttaacgcg
    10381 aattaattct gtggaatgtg tgtcagttag ggtgtggaaa gtccccaggc tccccagcag
    10441 gcagaagtat gcaaagcatg catctcaatt agtcagcaac caggtgtgga aagtccccag
    10501 gctccccagc aggcagaagt atgcaaagca tgcatctcaa ttagtcagca accatagtcc
    10561 cgcccctaac tccgcccagt tccgcccatt ctccgcccca tggctgacta atttttttta
    10621 tttatgcaga ggccgaggcc gcctctgcct ctgagctatt ccagaagtag tgaggaggct
    10681 tttttggagg cctaggcttt tgcaaaaagc tcccgggagc ttgtatatcc attttcggat
    10741 ctgatcagca cgtgttgaca attaatcatc ggcatagtat atcggcatag tataatacga
    10801 caaggtgagg aactaaacca tggccaagtt gaccagtgcc gttccggtgc tcaccgcgcg
    10861 cgacgtcgcc ggagcggtcg agttctggac cgaccggctc gggttctccc gggacttcgt
    10921 ggaggacgac ttcgccggtg tggtccggga cgacgtgacc ctgttcatca gcgcggtcca
    10981 ggaccaggtg gtgccggaca acaccctggc ctgggtgtgg gtgcgcggcc tggacgagct
    11041 gtacgccgag tggtcggagg tcgtgtccac gaacttccgg gacgcctccg ggccggccat
    11101 gaccgagatc ggcgagcagc cgtgggggcg ggagttcgcc ctgcgcgacc cggccggcaa
    11161 ctgcgtgcac ttcgtggccg aggagcagga ctgacacgtg ctacgagatt tcgattccac
    11221 cgccgccttc tatgaaaggt tgggcttcgg aatcgttttc cgggacgccg gctggatgat
    11281 cctccagcgc ggggatctca tgctggagtt cttcgcccac cccaacttgt ttattgcagc
    11341 ttataatggt tacaaataaa gcaatagcat cacaaatttc acaaataaag catttttttc
    11401 actgcattct agttgtggtt tgtccaaact catcaatgta tcttatcatg tctgtatacc
    11461 gtcgacctct agctagagct tggcgtaatc atggtcatag ctgtttcctg tgtgaaattg
    11521 ttatccgctc acaattccac acaacatacg agccggaagc ataaagtgta aagcctgggg
    11581 tgcctaatga gtgagctaac tcacattaat tgcgttgcgc tcactgcccg ctttccagtc
    11641 gggaaacctg tcgtgccagc tgcattaatg aatcggccaa cgcgcgggga gaggcggttt
    11701 gcgtattggg cgctcttccg cttcctcgct cactgactcg ctgcgctcgg tcgttcggct
    11761 gcggcgagcg gtatcagctc actcaaaggc ggtaatacgg ttatccacag aatcagggga
    11821 taacgcagga aagaacatgt gagcaaaagg ccagcaaaag gccaggaacc gtaaaaaggc
    11881 cgcgttgctg gcgtttttcc ataggctccg cccccctgac gagcatcaca aaaatcgacg
    11941 ctcaagtcag aggtggcgaa acccgacagg actataaaga taccaggcgt ttccccctgg
    12001 aagctccctc gtgcgctctc ctgttccgac cctgccgctt accggatacc tgtccgcctt
    12061 tctcccttcg ggaagcgtgg cgctttctca tagctcacgc tgtaggtatc tcagttcggt
    12121 gtaggtcgtt cgctccaagc tgggctgtgt gcacgaaccc cccgttcagc ccgaccgctg
    12181 cgccttatcc ggtaactatc gtcttgagtc caacccggta agacacgact tatcgccact
    12241 ggcagcagcc actggtaaca ggattagcag agcgaggtat gtaggcggtg ctacagagtt
    12301 cttgaagtgg tggcctaact acggctacac tagaagaaca gtatttggta tctgcgctct
    12361 gctgaagcca gttaccttcg gaaaaagagt tggtagctct tgatccggca aacaaaccac
    12421 cgctggtagc ggtggttttt ttgtttgcaa gcagcagatt acgcgcagaa aaaaaggatc
    12481 tcaagaagat cctttgatct tttctacggg gtctgacgct cagtggaacg aaaactcacg
    12541 ttaagggatt ttggtcatga gattatcaaa aaggatcttc acctagatcc ttttaaatta
    12601 aaaatgaagt tttaaatcaa tctaaagtat atatgagtaa acttggtctg acagttacca
    12661 atgcttaatc agtgaggcac ctatctcagc gatctgtcta tttcgttcat ccatagttgc
    12721 ctgactcccc gtcgtgtaga taactacgat acgggagggc ttaccatctg gccccagtgc
    12781 tgcaatgata ccgcgagacc cacgctcacc ggctccagat ttatcagcaa taaaccagcc
    12841 agccggaagg gccgagcgca gaagtggtcc tgcaacttta tccgcctcca tccagtctat
    12901 taattgttgc cgggaagcta gagtaagtag ttcgccagtt aatagtttgc gcaacgttgt
    12961 tgccattgct acaggcatcg tggtgtcacg ctcgtcgttt ggtatggctt cattcagctc
    13021 cggttcccaa cgatcaaggc gagttacatg atcccccatg ttgtgcaaaa aagcggttag
    13081 ctccttcggt cctccgatcg ttgtcagaag taagttggcc gcagtgttat cactcatggt
    13141 tatggcagca ctgcataatt ctcttactgt catgccatcc gtaagatgct tttctgtgac
    13201 tggtgagtac tcaaccaagt cattctgaga atagtgtatg cggcgaccga gttgctcttg
    13261 cccggcgtca atacgggata ataccgcgcc acatagcaga actttaaaag tgctcatcat
    13321 tggaaaacgt tcttcggggc gaaaactctc aaggatctta ccgctgttga gatccagttc
    13381 gatgtaaccc actcgtgcac ccaactgatc ttcagcatct tttactttca ccagcgtttc
    13441 tgggtgagca aaaacaggaa ggcaaaatgc cgcaaaaaag ggaataaggg cgacacggaa
    13501 atgttgaata ctcatactct tcctttttca atattattga agcatttatc agggttattg
    13561 tctcatgagc ggatacatat ttgaatgtat ttagaaaaat aaacaaatag gggttccgcg
    13621 cacatttccc cgaaaagtgc cacctgac
//
