LOCUS       sequence-339894-       13612 bp    DNA     circular SYN 15-DEC-2025
DEFINITION  Cas9 protein with BFP.
ACCESSION   .
VERSION     .
KEYWORDS    .
SOURCE      synthetic DNA construct
  ORGANISM  synthetic DNA construct
REFERENCE   1  (bases 1 to 13612)
  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 13612)
  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-BFP
FEATURES             Location/Qualifiers
     source          1..13612
                     /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"
     CDS             7137..7835
                     /codon_start=1
                     /product="monomeric blue fluorescent protein"
                     /label=TagBFP
                     /note="mammalian codon-optimized"
                     /translation="MSELIKENMHMKLYMEGTVDNHHFKCTSEGEGKPYEGTQTMRIKV
                     VEGGPLPFAFDILATSFLYGSKTFINHTQGIPDFFKQSFPEGFTWERVTTYEDGGVLTA
                     TQDTSLQDGCLIYNVKIRGVNFTSNGPVMQKKTLGWEAFTETLYPADGGLEGRNDMALK
                     LVGGSHLIANIKTTYRSKKPAKNLKMPGVYYVDYRLERIKEANNETYVEQHEVAVARYC
                     DLPSKLGHKLN"
     CDS             7845..7901
                     /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             7902..8300
                     /codon_start=1
                     /gene="Aspergillus terreus BSD"
                     /product="blasticidin S deaminase"
                     /label=BSD
                     /note="confers resistance to blasticidin"
                     /translation="MAKPLSQEESTLIERATATINSIPISEDYSVASAALSSDGRIFTG
                     VNVYHFTGGPCAELVVLGTAAAAAAGNLTCIVAIGNENRGILSPCGRCRQVLLDLHPGI
                     KAIVKDSDGQPTAVGIRELLPSGYVWEG"
     misc_feature    8325..8913
                     /label=WPRE
                     /note="woodchuck hepatitis virus posttranscriptional 
                     regulatory element"
     primer_bind     complement(8378..8398)
                     /label=WPRE-R
                     /note="WPRE, reverse primer"
     CDS             complement(8796..8807)
                     /codon_start=1
                     /product="Factor Xa recognition and cleavage site"
                     /label=Factor Xa site
                     /translation="IEGR"
     primer_bind     complement(8916..8932)
                     /label=KS primer
                     /note="common sequencing primer, one of multiple similar 
                     variants"
     primer_bind     complement(8917..8933)
                     /label=pBluescriptKS
                     /note="For pBluescript vector"
     LTR             9438..9618
                     /label=5' LTR (truncated)
                     /note="truncated 5' long terminal repeat (LTR) from HIV-1"
     primer_bind     complement(9644..9661)
                     /label=BGH-rev
                     /note="Bovine growth hormone terminator, reverse primer. 
                     Also called BGH reverse"
     polyA_signal    9650..9874
                     /label=bGH poly(A) signal
                     /note="bovine growth hormone polyadenylation signal"
     rep_origin      9920..10348
                     /direction=RIGHT
                     /label=f1 ori
                     /note="f1 bacteriophage origin of replication; arrow 
                     indicates direction of (+) strand synthesis"
     primer_bind     complement(10007..10026)
                     /label=F1ori-R
                     /note="F1 origin, reverse primer"
     primer_bind     10217..10238
                     /label=F1ori-F
                     /note="F1 origin, forward primer"
     primer_bind     complement(10357..10377)
                     /label=pBABE 3'
                     /note="SV40 enhancer, reverse primer for pBABE vectors"
     rep_origin      10522..10656
                     /label=SV40 ori
                     /note="SV40 origin of replication"
     primer_bind     10583..10602
                     /label=SV40pro-F
                     /note="SV40 promoter/origin, forward primer"
     promoter        10718..10765
                     /label=EM7 promoter
                     /note="synthetic bacterial promoter "
     CDS             10784..11158
                     /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    11288..11409
                     /label=SV40 poly(A) signal
                     /note="SV40 polyadenylation signal"
     primer_bind     complement(11325..11344)
                     /label=SV40pA-R
                     /note="SV40 polyA, reverse primer"
     primer_bind     11379..11398
                     /label=EBV-rev
                     /note="SV40 polyA terminator, reverse primer"
     primer_bind     complement(11458..11474)
                     /label=M13 rev
                     /note="common sequencing primer, one of multiple similar 
                     variants"
     primer_bind     complement(11458..11474)
                     /label=M13 Reverse
                     /note="In lacZ gene. Also called M13-rev"
     primer_bind     complement(11471..11493)
                     /label=M13/pUC Reverse
                     /note="In lacZ gene"
     protein_bind    11482..11498
                     /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(11506..11512,11513..11530,11531..11536))
                     /label=lac promoter
                     /note="promoter for the E. coli lac operon"
     protein_bind    11551..11572
                     /label=CAP binding site
                     /bound_moiety="E. coli catabolite activator protein"
                     /note="CAP binding activates transcription in the presence 
                     of cAMP."
     primer_bind     complement(11689..11706)
                     /label=L4440
                     /note="L4440 vector, forward primer"
     rep_origin      complement(11860..12448)
                     /direction=LEFT
                     /label=ori
                     /note="high-copy-number ColE1/pMB1/pBR322/pUC origin of 
                     replication"
     primer_bind     complement(11940..11959)
                     /label=pBR322ori-F
                     /note="pBR322 origin, forward primer"
     CDS             complement(join(12619..13410,13411..13479))
                     /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     13242..13261
                     /label=Amp-R
                     /note="Ampicillin resistance gene, reverse primer"
     promoter        complement(13480..13584)
                     /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 gccaccatga
     7141 gcgagctgat taaggagaac atgcacatga agctgtacat ggagggcacc gtggacaacc
     7201 atcacttcaa gtgcacatcc gagggcgaag gcaagcccta cgagggcacc cagaccatga
     7261 gaatcaaggt ggtcgagggc ggccctctcc ccttcgcctt cgacatcctg gctactagct
     7321 tcctctacgg cagcaagacc ttcatcaacc acacccaggg catccccgac ttcttcaagc
     7381 agtccttccc tgagggcttc acatgggaga gagtcaccac atacgaagac gggggcgtgc
     7441 tgaccgctac ccaggacacc agcctccagg acggctgcct catctacaac gtcaagatca
     7501 gaggggtgaa cttcacatcc aacggccctg tgatgcagaa gaaaacactc ggctgggagg
     7561 ccttcaccga gacgctgtac cccgctgacg gcggcctgga aggcagaaac gacatggccc
     7621 tgaagctcgt gggcgggagc catctgatcg caaacatcaa gaccacatat agatccaaga
     7681 aacccgctaa gaacctcaag atgcctggcg tctactatgt ggactacaga ctggaaagaa
     7741 tcaaggaggc caacaacgag acctacgtcg agcagcacga ggtggcagtg gccagatact
     7801 gcgacctccc tagcaaactg gggcacaagc ttaatggatc cggcgcaaca aacttctctc
     7861 tgctgaaaca agccggagat gtcgaagaga atcctggacc gatggccaag cctttgtctc
     7921 aagaagaatc caccctcatt gaaagagcaa cggctacaat caacagcatc cccatctctg
     7981 aagactacag cgtcgccagc gcagctctct ctagcgacgg ccgcatcttc actggtgtca
     8041 atgtatatca ttttactggg ggaccttgtg cagaactcgt ggtgctgggc actgctgctg
     8101 ctgcggcagc tggcaacctg acttgtatcg tcgcgatcgg aaatgagaac aggggcatct
     8161 tgagcccctg cggacggtgc cgacaggtgc ttctcgatct gcatcctggg atcaaagcca
     8221 tagtgaagga cagtgatgga cagccgacgg cagttgggat tcgtgaattg ctgccctctg
     8281 gttatgtgtg ggagggctaa gaattcgata tcaagcttat cggtaatcaa cctctggatt
     8341 acaaaatttg tgaaagattg actggtattc ttaactatgt tgctcctttt acgctatgtg
     8401 gatacgctgc tttaatgcct ttgtatcatg ctattgcttc ccgtatggct ttcattttct
     8461 cctccttgta taaatcctgg ttgctgtctc tttatgagga gttgtggccc gttgtcaggc
     8521 aacgtggcgt ggtgtgcact gtgtttgctg acgcaacccc cactggttgg ggcattgcca
     8581 ccacctgtca gctcctttcc gggactttcg ctttccccct ccctattgcc acggcggaac
     8641 tcatcgccgc ctgccttgcc cgctgctgga caggggctcg gctgttgggc actgacaatt
     8701 ccgtggtgtt gtcggggaaa tcatcgtcct ttccttggct gctcgcctgt gttgccacct
     8761 ggattctgcg cgggacgtcc ttctgctacg tcccttcggc cctcaatcca gcggaccttc
     8821 cttcccgcgg cctgctgccg gctctgcggc ctcttccgcg tcttcgcctt cgccctcaga
     8881 cgagtcggat ctccctttgg gccgcctccc cgcatcgata ccgtcgacct cgagacctag
     8941 aaaaacatgg agcaatcaca agtagcaata cagcagctac caatgctgat tgtgcctggc
     9001 tagaagcaca agaggaggag gaggtgggtt ttccagtcac acctcaggta cctttaagac
     9061 caatgactta caaggcagct gtagatctta gccacttttt aaaagaaaag gggggactgg
     9121 aagggctaat tcactcccaa cgaagacaag atatccttga tctgtggatc taccacacac
     9181 aaggctactt ccctgattgg cagaactaca caccagggcc agggatcaga tatccactga
     9241 cctttggatg gtgctacaag ctagtaccag ttgagcaaga gaaggtagaa gaagccaatg
     9301 aaggagagaa cacccgcttg ttacaccctg tgagcctgca tgggatggat gacccggaga
     9361 gagaagtatt agagtggagg tttgacagcc gcctagcatt tcatcacatg gcccgagagc
     9421 tgcatccgga ctgtactggg tctctctggt tagaccagat ctgagcctgg gagctctctg
     9481 gctaactagg gaacccactg cttaagcctc aataaagctt gccttgagtg cttcaagtag
     9541 tgtgtgcccg tctgttgtgt gactctggta actagagatc cctcagaccc ttttagtcag
     9601 tgtggaaaat ctctagcagg gcccgtttaa acccgctgat cagcctcgac tgtgccttct
     9661 agttgccagc catctgttgt ttgcccctcc cccgtgcctt ccttgaccct ggaaggtgcc
     9721 actcccactg tcctttccta ataaaatgag gaaattgcat cgcattgtct gagtaggtgt
     9781 cattctattc tggggggtgg ggtggggcag gacagcaagg gggaggattg ggaagacaat
     9841 agcaggcatg ctggggatgc ggtgggctct atggcttctg aggcggaaag aaccagctgg
     9901 ggctctaggg ggtatcccca cgcgccctgt agcggcgcat taagcgcggc gggtgtggtg
     9961 gttacgcgca gcgtgaccgc tacacttgcc agcgccctag cgcccgctcc tttcgctttc
    10021 ttcccttcct ttctcgccac gttcgccggc tttccccgtc aagctctaaa tcgggggctc
    10081 cctttagggt tccgatttag tgctttacgg cacctcgacc ccaaaaaact tgattagggt
    10141 gatggttcac gtagtgggcc atcgccctga tagacggttt ttcgcccttt gacgttggag
    10201 tccacgttct ttaatagtgg actcttgttc caaactggaa caacactcaa ccctatctcg
    10261 gtctattctt ttgatttata agggattttg ccgatttcgg cctattggtt aaaaaatgag
    10321 ctgatttaac aaaaatttaa cgcgaattaa ttctgtggaa tgtgtgtcag ttagggtgtg
    10381 gaaagtcccc aggctcccca gcaggcagaa gtatgcaaag catgcatctc aattagtcag
    10441 caaccaggtg tggaaagtcc ccaggctccc cagcaggcag aagtatgcaa agcatgcatc
    10501 tcaattagtc agcaaccata gtcccgcccc taactccgcc cagttccgcc cattctccgc
    10561 cccatggctg actaattttt tttatttatg cagaggccga ggccgcctct gcctctgagc
    10621 tattccagaa gtagtgagga ggcttttttg gaggcctagg cttttgcaaa aagctcccgg
    10681 gagcttgtat atccattttc ggatctgatc agcacgtgtt gacaattaat catcggcata
    10741 gtatatcggc atagtataat acgacaaggt gaggaactaa accatggcca agttgaccag
    10801 tgccgttccg gtgctcaccg cgcgcgacgt cgccggagcg gtcgagttct ggaccgaccg
    10861 gctcgggttc tcccgggact tcgtggagga cgacttcgcc ggtgtggtcc gggacgacgt
    10921 gaccctgttc atcagcgcgg tccaggacca ggtggtgccg gacaacaccc tggcctgggt
    10981 gtgggtgcgc ggcctggacg agctgtacgc cgagtggtcg gaggtcgtgt ccacgaactt
    11041 ccgggacgcc tccgggccgg ccatgaccga gatcggcgag cagccgtggg ggcgggagtt
    11101 cgccctgcgc gacccggccg gcaactgcgt gcacttcgtg gccgaggagc aggactgaca
    11161 cgtgctacga gatttcgatt ccaccgccgc cttctatgaa aggttgggct tcggaatcgt
    11221 tttccgggac gccggctgga tgatcctcca gcgcggggat ctcatgctgg agttcttcgc
    11281 ccaccccaac ttgtttattg cagcttataa tggttacaaa taaagcaata gcatcacaaa
    11341 tttcacaaat aaagcatttt tttcactgca ttctagttgt ggtttgtcca aactcatcaa
    11401 tgtatcttat catgtctgta taccgtcgac ctctagctag agcttggcgt aatcatggtc
    11461 atagctgttt cctgtgtgaa attgttatcc gctcacaatt ccacacaaca tacgagccgg
    11521 aagcataaag tgtaaagcct ggggtgccta atgagtgagc taactcacat taattgcgtt
    11581 gcgctcactg cccgctttcc agtcgggaaa cctgtcgtgc cagctgcatt aatgaatcgg
    11641 ccaacgcgcg gggagaggcg gtttgcgtat tgggcgctct tccgcttcct cgctcactga
    11701 ctcgctgcgc tcggtcgttc ggctgcggcg agcggtatca gctcactcaa aggcggtaat
    11761 acggttatcc acagaatcag gggataacgc aggaaagaac atgtgagcaa aaggccagca
    11821 aaaggccagg aaccgtaaaa aggccgcgtt gctggcgttt ttccataggc tccgcccccc
    11881 tgacgagcat cacaaaaatc gacgctcaag tcagaggtgg cgaaacccga caggactata
    11941 aagataccag gcgtttcccc ctggaagctc cctcgtgcgc tctcctgttc cgaccctgcc
    12001 gcttaccgga tacctgtccg cctttctccc ttcgggaagc gtggcgcttt ctcatagctc
    12061 acgctgtagg tatctcagtt cggtgtaggt cgttcgctcc aagctgggct gtgtgcacga
    12121 accccccgtt cagcccgacc gctgcgcctt atccggtaac tatcgtcttg agtccaaccc
    12181 ggtaagacac gacttatcgc cactggcagc agccactggt aacaggatta gcagagcgag
    12241 gtatgtaggc ggtgctacag agttcttgaa gtggtggcct aactacggct acactagaag
    12301 aacagtattt ggtatctgcg ctctgctgaa gccagttacc ttcggaaaaa gagttggtag
    12361 ctcttgatcc ggcaaacaaa ccaccgctgg tagcggtggt ttttttgttt gcaagcagca
    12421 gattacgcgc agaaaaaaag gatctcaaga agatcctttg atcttttcta cggggtctga
    12481 cgctcagtgg aacgaaaact cacgttaagg gattttggtc atgagattat caaaaaggat
    12541 cttcacctag atccttttaa attaaaaatg aagttttaaa tcaatctaaa gtatatatga
    12601 gtaaacttgg tctgacagtt accaatgctt aatcagtgag gcacctatct cagcgatctg
    12661 tctatttcgt tcatccatag ttgcctgact ccccgtcgtg tagataacta cgatacggga
    12721 gggcttacca tctggcccca gtgctgcaat gataccgcga gacccacgct caccggctcc
    12781 agatttatca gcaataaacc agccagccgg aagggccgag cgcagaagtg gtcctgcaac
    12841 tttatccgcc tccatccagt ctattaattg ttgccgggaa gctagagtaa gtagttcgcc
    12901 agttaatagt ttgcgcaacg ttgttgccat tgctacaggc atcgtggtgt cacgctcgtc
    12961 gtttggtatg gcttcattca gctccggttc ccaacgatca aggcgagtta catgatcccc
    13021 catgttgtgc aaaaaagcgg ttagctcctt cggtcctccg atcgttgtca gaagtaagtt
    13081 ggccgcagtg ttatcactca tggttatggc agcactgcat aattctctta ctgtcatgcc
    13141 atccgtaaga tgcttttctg tgactggtga gtactcaacc aagtcattct gagaatagtg
    13201 tatgcggcga ccgagttgct cttgcccggc gtcaatacgg gataataccg cgccacatag
    13261 cagaacttta aaagtgctca tcattggaaa acgttcttcg gggcgaaaac tctcaaggat
    13321 cttaccgctg ttgagatcca gttcgatgta acccactcgt gcacccaact gatcttcagc
    13381 atcttttact ttcaccagcg tttctgggtg agcaaaaaca ggaaggcaaa atgccgcaaa
    13441 aaagggaata agggcgacac ggaaatgttg aatactcata ctcttccttt ttcaatatta
    13501 ttgaagcatt tatcagggtt attgtctcat gagcggatac atatttgaat gtatttagaa
    13561 aaataaacaa ataggggttc cgcgcacatt tccccgaaaa gtgccacctg ac
//
