LOCUS       sequence-231696-       10711 bp    DNA     circular SYN 15-DEC-2025
DEFINITION  Encodes Cas9, T2A-mCherry, and AmpR which creates a CRISPR system 
            application with reliable positive selection.
ACCESSION   .
VERSION     .
KEYWORDS    .
SOURCE      synthetic DNA construct
  ORGANISM  synthetic DNA construct
REFERENCE   1  (bases 1 to 10711)
  TITLE     CRISPR-Cas9 system with SacB toxin Positive Selection
  JOURNAL   Unpublished
REFERENCE   2  (bases 1 to 10711)
  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: 
            "Unpublished"
COMMENT     Sequence Label: pU6-SacB-Cas9-T2A-mCherry_(BbsI)
FEATURES             Location/Qualifiers
     source          1..10711
                     /mol_type="other DNA"
                     /organism="synthetic DNA construct"
     promoter        1..249
                     /label=U6 promoter
                     /note="RNA polymerase III promoter for human U6 snRNA 
                     (Domitrovich & Kunkel, 2003)"
     primer_bind     1..21
                     /label=hU6-F
                     /note="Human U6 promoter, forward primer"
     primer_bind     172..191
                     /label=LKO.1 5'
                     /note="Human U6 promoter, forward primer"
     CDS             join(273..359,360..1694)
                     /codon_start=1
                     /gene="Bacillus subtilis sacB"
                     /product="secreted levansucrase that renders bacterial 
                     growth sensitive to sucrose"
                     /label=SacB
                     /note="negative selection marker"
                     /translation="MNIKKFAKQATVLTFTTALLAGGATQAFAKETNQKPYKETYGISH
                     ITRHDMLQIPEQQKNEKYQVPEFDSSTIKNISSAKGLDVWDSWPLQNADGTVANYHGYH
                     IVFALAGDPKNADDTSIYMFYQKVGETSIDSWKNAGRVFKDSDKFDANDSILKDQTQEW
                     SGSATFTSDGKIRLFYTDFSGKHYGKQTLTTAQVNVSASDSSLNINGVEDYKSIFDGDG
                     KTYQNVQQFIDEGNYSSGDNHTLRDPHYVEDKGHKYLVFEANTGTEDGYQGEESLFNKA
                     YYGKSTSFFRQESQKLLQSDKKRTAELANGALGMIELNDDYTLKKVMKPLIASNTVTDE
                     IERANVFKMNGKWYLFTDSRGSKMTIDGITSNDIYMLGYVSNSLTGPYKPLNKTGLVLK
                     MDLDPNDVTFTYSHFAVPQAKGNNVVITSYMTNRGFYADKQSTFAPSFLLNIKGKKTSV
                     VKDSILEQGQLTVNK"
     misc_RNA        1703..1778
                     /label=gRNA scaffold
                     /note="guide RNA scaffold for the Streptococcus pyogenes 
                     CRISPR/Cas9 system"
     enhancer        1875..2160
                     /label=CMV enhancer
                     /note="human cytomegalovirus immediate early enhancer; 
                     contains an 18-bp deletion relative to the standard CMV 
                     enhancer"
     promoter        2162..2439
                     /label=chicken beta-actin promoter
     intron          2440..2667
                     /label=hybrid intron
                     /note="hybrid between chicken beta-actin (CBA) and minute 
                     virus of mice (MMV) introns (Gray et al., 2011)"
     regulatory      2679..2688
                     /label=Kozak sequence
                     /note="vertebrate consensus sequence for strong initiation 
                     of translation (Kozak, 1987)"
                     /regulatory_class="other"
     CDS             2688..2753
                     /codon_start=1
                     /product="three tandem FLAG(R) epitope tags, followed by an
                     enterokinase cleavage site"
                     /label=3xFLAG
                     /translation="DYKDHDGDYKDHDIDYKDDDDK"
     CDS             2760..2780
                     /codon_start=1
                     /product="nuclear localization signal of SV40 (simian virus
                     40) large T antigen"
                     /label=SV40 NLS
                     /translation="PKKKRKV"
     CDS             2805..6905
                     /codon_start=1
                     /product="Cas9 (Csn1) endonuclease from the Streptococcus 
                     pyogenes Type II CRISPR/Cas system"
                     /label=Cas9
                     /note="generates RNA-guided double strand breaks in DNA"
                     /translation="DKKYSIGLDIGTNSVGWAVITDEYKVPSKKFKVLGNTDRHSIKKN
                     LIGALLFDSGETAEATRLKRTARRRYTRRKNRICYLQEIFSNEMAKVDDSFFHRLEESF
                     LVEEDKKHERHPIFGNIVDEVAYHEKYPTIYHLRKKLVDSTDKADLRLIYLALAHMIKF
                     RGHFLIEGDLNPDNSDVDKLFIQLVQTYNQLFEENPINASGVDAKAILSARLSKSRRLE
                     NLIAQLPGEKKNGLFGNLIALSLGLTPNFKSNFDLAEDAKLQLSKDTYDDDLDNLLAQI
                     GDQYADLFLAAKNLSDAILLSDILRVNTEITKAPLSASMIKRYDEHHQDLTLLKALVRQ
                     QLPEKYKEIFFDQSKNGYAGYIDGGASQEEFYKFIKPILEKMDGTEELLVKLNREDLLR
                     KQRTFDNGSIPHQIHLGELHAILRRQEDFYPFLKDNREKIEKILTFRIPYYVGPLARGN
                     SRFAWMTRKSEETITPWNFEEVVDKGASAQSFIERMTNFDKNLPNEKVLPKHSLLYEYF
                     TVYNELTKVKYVTEGMRKPAFLSGEQKKAIVDLLFKTNRKVTVKQLKEDYFKKIECFDS
                     VEISGVEDRFNASLGTYHDLLKIIKDKDFLDNEENEDILEDIVLTLTLFEDREMIEERL
                     KTYAHLFDDKVMKQLKRRRYTGWGRLSRKLINGIRDKQSGKTILDFLKSDGFANRNFMQ
                     LIHDDSLTFKEDIQKAQVSGQGDSLHEHIANLAGSPAIKKGILQTVKVVDELVKVMGRH
                     KPENIVIEMARENQTTQKGQKNSRERMKRIEEGIKELGSQILKEHPVENTQLQNEKLYL
                     YYLQNGRDMYVDQELDINRLSDYDVDHIVPQSFLKDDSIDNKVLTRSDKNRGKSDNVPS
                     EEVVKKMKNYWRQLLNAKLITQRKFDNLTKAERGGLSELDKAGFIKRQLVETRQITKHV
                     AQILDSRMNTKYDENDKLIREVKVITLKSKLVSDFRKDFQFYKVREINNYHHAHDAYLN
                     AVVGTALIKKYPKLESEFVYGDYKVYDVRKMIAKSEQEIGKATAKYFFYSNIMNFFKTE
                     ITLANGEIRKRPLIETNGETGEIVWDKGRDFATVRKVLSMPQVNIVKKTEVQTGGFSKE
                     SILPKRNSDKLIARKKDWDPKKYGGFDSPTVAYSVLVVAKVEKGKSKKLKSVKELLGIT
                     IMERSSFEKNPIDFLEAKGYKEVKKDLIIKLPKYSLFELENGRKRMLASAGELQKGNEL
                     ALPSKYVNFLYLASHYEKLKGSPEDNEQKQLFVEQHKHYLDEIIEQISEFSKRVILADA
                     NLDKVLSAYNKHRDKPIREQAENIIHLFTLTNLGAPAAFKYFDTTIDRKRYTSTKEVLD
                     ATLIHQSITGLYETRIDLSQLGGD"
     CDS             2805..6905
                     /codon_start=1
                     /product="Cas9 (Csn1) endonuclease from the Streptococcus 
                     pyogenes Type II CRISPR/Cas system"
                     /label=Cas9
                     /note="generates RNA-guided double strand breaks in DNA"
                     /translation="DKKYSIGLDIGTNSVGWAVITDEYKVPSKKFKVLGNTDRHSIKKN
                     LIGALLFDSGETAEATRLKRTARRRYTRRKNRICYLQEIFSNEMAKVDDSFFHRLEESF
                     LVEEDKKHERHPIFGNIVDEVAYHEKYPTIYHLRKKLVDSTDKADLRLIYLALAHMIKF
                     RGHFLIEGDLNPDNSDVDKLFIQLVQTYNQLFEENPINASGVDAKAILSARLSKSRRLE
                     NLIAQLPGEKKNGLFGNLIALSLGLTPNFKSNFDLAEDAKLQLSKDTYDDDLDNLLAQI
                     GDQYADLFLAAKNLSDAILLSDILRVNTEITKAPLSASMIKRYDEHHQDLTLLKALVRQ
                     QLPEKYKEIFFDQSKNGYAGYIDGGASQEEFYKFIKPILEKMDGTEELLVKLNREDLLR
                     KQRTFDNGSIPHQIHLGELHAILRRQEDFYPFLKDNREKIEKILTFRIPYYVGPLARGN
                     SRFAWMTRKSEETITPWNFEEVVDKGASAQSFIERMTNFDKNLPNEKVLPKHSLLYEYF
                     TVYNELTKVKYVTEGMRKPAFLSGEQKKAIVDLLFKTNRKVTVKQLKEDYFKKIECFDS
                     VEISGVEDRFNASLGTYHDLLKIIKDKDFLDNEENEDILEDIVLTLTLFEDREMIEERL
                     KTYAHLFDDKVMKQLKRRRYTGWGRLSRKLINGIRDKQSGKTILDFLKSDGFANRNFMQ
                     LIHDDSLTFKEDIQKAQVSGQGDSLHEHIANLAGSPAIKKGILQTVKVVDELVKVMGRH
                     KPENIVIEMARENQTTQKGQKNSRERMKRIEEGIKELGSQILKEHPVENTQLQNEKLYL
                     YYLQNGRDMYVDQELDINRLSDYDVDHIVPQSFLKDDSIDNKVLTRSDKNRGKSDNVPS
                     EEVVKKMKNYWRQLLNAKLITQRKFDNLTKAERGGLSELDKAGFIKRQLVETRQITKHV
                     AQILDSRMNTKYDENDKLIREVKVITLKSKLVSDFRKDFQFYKVREINNYHHAHDAYLN
                     AVVGTALIKKYPKLESEFVYGDYKVYDVRKMIAKSEQEIGKATAKYFFYSNIMNFFKTE
                     ITLANGEIRKRPLIETNGETGEIVWDKGRDFATVRKVLSMPQVNIVKKTEVQTGGFSKE
                     SILPKRNSDKLIARKKDWDPKKYGGFDSPTVAYSVLVVAKVEKGKSKKLKSVKELLGIT
                     IMERSSFEKNPIDFLEAKGYKEVKKDLIIKLPKYSLFELENGRKRMLASAGELQKGNEL
                     ALPSKYVNFLYLASHYEKLKGSPEDNEQKQLFVEQHKHYLDEIIEQISEFSKRVILADA
                     NLDKVLSAYNKHRDKPIREQAENIIHLFTLTNLGAPAAFKYFDTTIDRKRYTSTKEVLD
                     ATLIHQSITGLYETRIDLSQLGGD"
     CDS             6906..6953
                     /codon_start=1
                     /product="bipartite nuclear localization signal from 
                     nucleoplasmin"
                     /label=nucleoplasmin NLS
                     /translation="KRPAATKKAGQAKKKK"
     CDS             6957..7010
                     /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             7017..7727
                     /codon_start=1
                     /product="monomeric derivative of DsRed fluorescent protein
                     (Shaner et al., 2004)"
                     /label=mCherry
                     /note="mammalian codon-optimized"
                     /translation="MVSKGEEDNMAIIKEFMRFKVHMEGSVNGHEFEIEGEGEGRPYEG
                     TQTAKLKVTKGGPLPFAWDILSPQFMYGSKAYVKHPADIPDYLKLSFPEGFKWERVMNF
                     EDGGVVTVTQDSSLQDGEFIYKVKLRGTNFPSDGPVMQKKTMGWEASSERMYPEDGALK
                     GEIKQRLKLKDGGHYDAEVKTTYKAKKPVQLPGAYNVNIKLDITSHNEDYTIVEQYERA
                     EGRHSTGGMDELYK"
     primer_bind     complement(7162..7180)
                     /label=mCherry-R
                     /note="mCherry, reverse primer"
     primer_bind     complement(7752..7769)
                     /label=BGH-rev
                     /note="Bovine growth hormone terminator, reverse primer. 
                     Also called BGH reverse"
     polyA_signal    7758..7965
                     /label=bGH poly(A) signal
                     /note="bovine growth hormone polyadenylation signal"
     repeat_region   7974..8114
                     /label=AAV2 ITR
                     /note="inverted terminal repeat of adeno-associated virus 
                     serotype 2"
     repeat_region   7974..8103
                     /label=AAV2 ITR
     rep_origin      8189..8644
                     /direction=RIGHT
                     /label=f1 ori
                     /note="f1 bacteriophage origin of replication; arrow 
                     indicates direction of (+) strand synthesis"
     primer_bind     complement(8276..8295)
                     /label=F1ori-R
                     /note="F1 origin, reverse primer"
     primer_bind     8486..8507
                     /label=F1ori-F
                     /note="F1 origin, forward primer"
     primer_bind     complement(8661..8680)
                     /label=pRS-marker
                     /note="pRS vectors, use to sequence yeast selectable 
                     marker"
     primer_bind     8780..8802
                     /label=pGEX 3'
                     /note="pGEX vectors, reverse primer"
     primer_bind     complement(8840..8858)
                     /label=pBRforEco
                     /note="pBR322 vectors, upsteam of EcoRI site, forward 
                     primer"
     promoter        8926..9030
                     /gene="bla"
                     /label=AmpR promoter
     CDS             join(9031..9099,9100..9891)
                     /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     complement(9249..9268)
                     /label=Amp-R
                     /note="Ampicillin resistance gene, reverse primer"
     rep_origin      10062..10650
                     /direction=RIGHT
                     /label=ori
                     /note="high-copy-number ColE1/pMB1/pBR322/pUC origin of 
                     replication"
     primer_bind     10551..10570
                     /label=pBR322ori-F
                     /note="pBR322 origin, forward primer"
ORIGIN
        1 gagggcctat ttcccatgat tccttcatat ttgcatatac gatacaaggc tgttagagag
       61 ataattggaa ttaatttgac tgtaaacaca aagatattag tacaaaatac gtgacgtaga
      121 aagtaataat ttcttgggta gtttgcagtt ttaaaattat gttttaaaat ggactatcat
      181 atgcttaccg taacttgaaa gtatttcgat ttcttggctt tatatatctt gtggaaagga
      241 cgaaacaccg ggtcttcaag gagacatgaa cgatgaacat caaaaagttt gcaaaacaag
      301 caacagtatt aacctttact accgcactgc tggcaggagg cgcaactcaa gcgtttgcga
      361 aagaaacgaa ccaaaagcca tataaggaaa catacggcat ttcccatatt acacgccatg
      421 atatgctgca aatccctgaa cagcaaaaaa atgaaaaata tcaagttcct gaattcgatt
      481 cgtccacaat taaaaatatc tcttctgcaa aaggcctgga cgtttgggac agctggccat
      541 tacaaaacgc tgacggcact gtcgcaaact atcacggcta ccacatcgtc tttgcattag
      601 ccggagatcc taaaaatgcg gatgacacat cgatttacat gttctatcaa aaagtcggcg
      661 aaacttctat tgacagctgg aaaaacgctg gccgcgtctt taaagacagc gacaaattcg
      721 atgcaaatga ttctatccta aaagaccaaa cacaagaatg gtcaggttca gccacattta
      781 catctgacgg aaaaatccgt ttattctaca ctgatttctc cggtaaacat tacggcaaac
      841 aaacactgac aactgcacaa gttaacgtat cagcatcaga cagctctttg aacatcaacg
      901 gtgtagagga ttataaatca atctttgacg gtgacggaaa aacgtatcaa aatgtacagc
      961 agttcatcga tgaaggcaac tacagctcag gcgacaacca tacgctgaga gatcctcact
     1021 acgtagaaga taaaggccac aaatacttag tatttgaagc aaacactgga actgaagatg
     1081 gctaccaagg cgaagaatct ttatttaaca aagcatacta tggcaaaagc acatcattct
     1141 tccgtcaaga aagtcaaaaa cttctgcaaa gcgataaaaa acgcacggct gagttagcaa
     1201 acggcgctct cggtatgatt gagctaaacg atgattacac actgaaaaaa gtgatgaaac
     1261 cgctgattgc atctaacaca gtaacagatg aaattgaacg cgcgaacgtc tttaaaatga
     1321 acggcaaatg gtacctgttc actgactccc gcggatcaaa aatgacgatt gacggcatta
     1381 cgtctaacga tatttacatg cttggttatg tttctaattc tttaactggc ccatacaagc
     1441 cgctgaacaa aactggcctt gtgttaaaaa tggatcttga tcctaacgat gtaaccttta
     1501 cttactcaca cttcgctgta cctcaagcga aaggaaacaa tgtcgtgatt acaagctata
     1561 tgacaaacag aggattctac gcagacaaac aatcaacgtt tgcgccaagc ttcctgctga
     1621 acatcaaagg caagaaaaca tctgttgtca aagacagcat ccttgaacaa ggacaattaa
     1681 cagttaacaa ataagaagac ctgttttaga gctagaaata gcaagttaaa ataaggctag
     1741 tccgttatca acttgaaaaa gtggcaccga gtcggtgctt ttttgtttta gagctagaaa
     1801 tagcaagtta aaataaggct agtccgtttt tagcgcgtgc gccaattctg cagacaaatg
     1861 gctctagagg tacccgttac ataacttacg gtaaatggcc cgcctggctg accgcccaac
     1921 gacccccgcc cattgacgtc aatagtaacg ccaataggga ctttccattg acgtcaatgg
     1981 gtggagtatt tacggtaaac tgcccacttg gcagtacatc aagtgtatca tatgccaagt
     2041 acgcccccta ttgacgtcaa tgacggtaaa tggcccgcct ggcattgtgc ccagtacatg
     2101 accttatggg actttcctac ttggcagtac atctacgtat tagtcatcgc tattaccatg
     2161 gtcgaggtga gccccacgtt ctgcttcact ctccccatct cccccccctc cccaccccca
     2221 attttgtatt tatttatttt ttaattattt tgtgcagcga tgggggcggg gggggggggg
     2281 gggcgcgcgc crggsggggs ggggsggggs grggggsggg gsggggcgag gcggagaggt
     2341 gcggcggcag ccaatcagag cggcgcgctc cgaaagtttc cttttatggc gaggcggcgg
     2401 cggcggcggc cctataaaaa gcgaagcgcg cggcgggcgg gagtcgctgc gcgctgcctt
     2461 cgccccgtgc cccgctccgc cgccgcctcg cgccgcccgc cccggctctg actgaccgcg
     2521 ttactcccac aggtgagcgg gcgggacggc ccttctcctc cgggctgtaa ttagctgagc
     2581 aagaggtaag ggtttaaggg atggttggtt ggtggggtat taatgtttaa ttacctggag
     2641 cacctgcctg aaatcacttt ttttcaggtt ggaccggtgc caccatggac tataaggacc
     2701 acgacggaga ctacaaggat catgatattg attacaaaga cgatgacgat aagatggccc
     2761 caaagaagaa gcggaaggtc ggtatccacg gagtcccagc agccgacaag aagtacagca
     2821 tcggcctgga catcggcacc aactctgtgg gctgggccgt gatcaccgac gagtacaagg
     2881 tgcccagcaa gaaattcaag gtgctgggca acaccgaccg gcacagcatc aagaagaacc
     2941 tgatcggagc cctgctgttc gacagcggcg aaacagccga ggccacccgg ctgaagagaa
     3001 ccgccagaag aagatacacc agacggaaga accggatctg ctatctgcaa gagatcttca
     3061 gcaacgagat ggccaaggtg gacgacagct tcttccacag actggaagag tccttcctgg
     3121 tggaagagga taagaagcac gagcggcacc ccatcttcgg caacatcgtg gacgaggtgg
     3181 cctaccacga gaagtacccc accatctacc acctgagaaa gaaactggtg gacagcaccg
     3241 acaaggccga cctgcggctg atctatctgg ccctggccca catgatcaag ttccggggcc
     3301 acttcctgat cgagggcgac ctgaaccccg acaacagcga cgtggacaag ctgttcatcc
     3361 agctggtgca gacctacaac cagctgttcg aggaaaaccc catcaacgcc agcggcgtgg
     3421 acgccaaggc catcctgtct gccagactga gcaagagcag acggctggaa aatctgatcg
     3481 cccagctgcc cggcgagaag aagaatggcc tgttcggaaa cctgattgcc ctgagcctgg
     3541 gcctgacccc caacttcaag agcaacttcg acctggccga ggatgccaaa ctgcagctga
     3601 gcaaggacac ctacgacgac gacctggaca acctgctggc ccagatcggc gaccagtacg
     3661 ccgacctgtt tctggccgcc aagaacctgt ccgacgccat cctgctgagc gacatcctga
     3721 gagtgaacac cgagatcacc aaggcccccc tgagcgcctc tatgatcaag agatacgacg
     3781 agcaccacca ggacctgacc ctgctgaaag ctctcgtgcg gcagcagctg cctgagaagt
     3841 acaaagagat tttcttcgac cagagcaaga acggctacgc cggctacatt gacggcggag
     3901 ccagccagga agagttctac aagttcatca agcccatcct ggaaaagatg gacggcaccg
     3961 aggaactgct cgtgaagctg aacagagagg acctgctgcg gaagcagcgg accttcgaca
     4021 acggcagcat cccccaccag atccacctgg gagagctgca cgccattctg cggcggcagg
     4081 aagattttta cccattcctg aaggacaacc gggaaaagat cgagaagatc ctgaccttcc
     4141 gcatccccta ctacgtgggc cctctggcca ggggaaacag cagattcgcc tggatgacca
     4201 gaaagagcga ggaaaccatc accccctgga acttcgagga agtggtggac aagggcgctt
     4261 ccgcccagag cttcatcgag cggatgacca acttcgataa gaacctgccc aacgagaagg
     4321 tgctgcccaa gcacagcctg ctgtacgagt acttcaccgt gtataacgag ctgaccaaag
     4381 tgaaatacgt gaccgaggga atgagaaagc ccgccttcct gagcggcgag cagaaaaagg
     4441 ccatcgtgga cctgctgttc aagaccaacc ggaaagtgac cgtgaagcag ctgaaagagg
     4501 actacttcaa gaaaatcgag tgcttcgact ccgtggaaat ctccggcgtg gaagatcggt
     4561 tcaacgcctc cctgggcaca taccacgatc tgctgaaaat tatcaaggac aaggacttcc
     4621 tggacaatga ggaaaacgag gacattctgg aagatatcgt gctgaccctg acactgtttg
     4681 aggacagaga gatgatcgag gaacggctga aaacctatgc ccacctgttc gacgacaaag
     4741 tgatgaagca gctgaagcgg cggagataca ccggctgggg caggctgagc cggaagctga
     4801 tcaacggcat ccgggacaag cagtccggca agacaatcct ggatttcctg aagtccgacg
     4861 gcttcgccaa cagaaacttc atgcagctga tccacgacga cagcctgacc tttaaagagg
     4921 acatccagaa agcccaggtg tccggccagg gcgatagcct gcacgagcac attgccaatc
     4981 tggccggcag ccccgccatt aagaagggca tcctgcagac agtgaaggtg gtggacgagc
     5041 tcgtgaaagt gatgggccgg cacaagcccg agaacatcgt gatcgaaatg gccagagaga
     5101 accagaccac ccagaaggga cagaagaaca gccgcgagag aatgaagcgg atcgaagagg
     5161 gcatcaaaga gctgggcagc cagatcctga aagaacaccc cgtggaaaac acccagctgc
     5221 agaacgagaa gctgtacctg tactacctgc agaatgggcg ggatatgtac gtggaccagg
     5281 aactggacat caaccggctg tccgactacg atgtggacca tatcgtgcct cagagctttc
     5341 tgaaggacga ctccatcgac aacaaggtgc tgaccagaag cgacaagaac cggggcaaga
     5401 gcgacaacgt gccctccgaa gaggtcgtga agaagatgaa gaactactgg cggcagctgc
     5461 tgaacgccaa gctgattacc cagagaaagt tcgacaatct gaccaaggcc gagagaggcg
     5521 gcctgagcga actggataag gccggcttca tcaagagaca gctggtggaa acccggcaga
     5581 tcacaaagca cgtggcacag atcctggact cccggatgaa cactaagtac gacgagaatg
     5641 acaagctgat ccgggaagtg aaagtgatca ccctgaagtc caagctggtg tccgatttcc
     5701 ggaaggattt ccagttttac aaagtgcgcg agatcaacaa ctaccaccac gcccacgacg
     5761 cctacctgaa cgccgtcgtg ggaaccgccc tgatcaaaaa gtaccctaag ctggaaagcg
     5821 agttcgtgta cggcgactac aaggtgtacg acgtgcggaa gatgatcgcc aagagcgagc
     5881 aggaaatcgg caaggctacc gccaagtact tcttctacag caacatcatg aactttttca
     5941 agaccgagat taccctggcc aacggcgaga tccggaagcg gcctctgatc gagacaaacg
     6001 gcgaaaccgg ggagatcgtg tgggataagg gccgggattt tgccaccgtg cggaaagtgc
     6061 tgagcatgcc ccaagtgaat atcgtgaaaa agaccgaggt gcagacaggc ggcttcagca
     6121 aagagtctat cctgcccaag aggaacagcg ataagctgat cgccagaaag aaggactggg
     6181 accctaagaa gtacggcggc ttcgacagcc ccaccgtggc ctattctgtg ctggtggtgg
     6241 ccaaagtgga aaagggcaag tccaagaaac tgaagagtgt gaaagagctg ctggggatca
     6301 ccatcatgga aagaagcagc ttcgagaaga atcccatcga ctttctggaa gccaagggct
     6361 acaaagaagt gaaaaaggac ctgatcatca agctgcctaa gtactccctg ttcgagctgg
     6421 aaaacggccg gaagagaatg ctggcctctg ccggcgaact gcagaaggga aacgaactgg
     6481 ccctgccctc caaatatgtg aacttcctgt acctggccag ccactatgag aagctgaagg
     6541 gctcccccga ggataatgag cagaaacagc tgtttgtgga acagcacaag cactacctgg
     6601 acgagatcat cgagcagatc agcgagttct ccaagagagt gatcctggcc gacgctaatc
     6661 tggacaaagt gctgtccgcc tacaacaagc accgggataa gcccatcaga gagcaggccg
     6721 agaatatcat ccacctgttt accctgacca atctgggagc ccctgccgcc ttcaagtact
     6781 ttgacaccac catcgaccgg aagaggtaca ccagcaccaa agaggtgctg gacgccaccc
     6841 tgatccacca gagcatcacc ggcctgtacg agacacggat cgacctgtct cagctgggag
     6901 gcgacaaaag gccggcggcc acgaaaaagg ccggccaggc aaaaaagaaa aagcttgagg
     6961 gcagaggaag tctgctaaca tgcggtgacg tggaggagaa tcccggccct gctagcatgg
     7021 tgagcaaggg cgaggaggat aacatggcca tcatcaagga gttcatgcgc ttcaaggtgc
     7081 acatggaggg ctccgtgaac ggccacgagt tcgagatcga gggcgagggc gagggccgcc
     7141 cctacgaggg cacccagacc gccaagctga aggtgaccaa gggtggcccc ctgcccttcg
     7201 cctgggacat cctgtcccct cagttcatgt acggctccaa ggcctacgtg aagcaccccg
     7261 ccgacatccc cgactacttg aagctgtcct tccccgaggg cttcaagtgg gagcgcgtga
     7321 tgaacttcga ggacggcggc gtggtgaccg tgacccagga ctcctccctg caggacggcg
     7381 agttcatcta caaggtgaag ctgcgcggca ccaacttccc ctcagacggc cccgtaatgc
     7441 agaagaaaac catgggctgg gaggcctcct ccgagcggat gtaccccgag gacggcgccc
     7501 tgaagggcga gatcaagcag aggctgaagc tgaaggacgg cggccactac gacgctgagg
     7561 tcaagaccac ctacaaggcc aagaagcccg tgcagctgcc cggcgcctac aacgtcaaca
     7621 tcaagttgga catcacctcc cacaacgagg actacaccat cgtggaacag tacgaacgcg
     7681 ccgagggccg ccactccacc ggcggcatgg acgagctgta caagtaagaa ttcctagagc
     7741 tcgctgatca gcctcgactg tgccttctag ttgccagcca tctgttgttt gcccctcccc
     7801 cgtgccttcc ttgaccctgg aaggtgccac tcccactgtc ctttcctaat aaaatgagga
     7861 aattgcatcg cattgtctga gtaggtgtca ttctattctg gggggtgggg tggggcagga
     7921 cagcaagggg gaggattggg aagagaatag caggcatgct ggggagcggc cgcaggaacc
     7981 cctagtgatg gagttggcca ctccctctct gcgcgctcgc tcgctcactg aggccgggcg
     8041 accaaaggtc gcccgacgcc cgggctttgc ccgggcggcc tcagtgagcg agcgagcgcg
     8101 cagctgcctg caggggcgcc tgatgcggta ttttctcctt acgcatctgt gcggtatttc
     8161 acaccgcata cgtcaaagca accatagtac gcgccctgta gcggcgcatt aagcgcggcg
     8221 ggtgtggtgg ttacgcgcag cgtgaccgct acacttgcca gcgccttagc gcccgctcct
     8281 ttcgctttct tcccttcctt tctcgccacg ttcgccggct ttccccgtca agctctaaat
     8341 cgggggctcc ctttagggtt ccgatttagt gctttacggc acctcgaccc caaaaaactt
     8401 gatttgggtg atggttcacg tagtgggcca tcgccctgat agacggtttt tcgccctttg
     8461 acgttggagt ccacgttctt taatagtgga ctcttgttcc aaactggaac aacactcaac
     8521 tctatctcgg gctattcttt tgatttataa gggattttgc cgatttcggt ctattggtta
     8581 aaaaatgagc tgatttaaca aaaatttaac gcgaatttta acaaaatatt aacgtttaca
     8641 attttatggt gcactctcag tacaatctgc tctgatgccg catagttaag ccagccccga
     8701 cacccgccaa cacccgctga cgcgccctga cgggcttgtc tgctcccggc atccgcttac
     8761 agacaagctg tgaccgtctc cgggagctgc atgtgtcaga ggttttcacc gtcatcaccg
     8821 aaacgcgcga gacgaaaggg cctcgtgata cgcctatttt tataggttaa tgtcatgata
     8881 ataatggttt cttagacgtc aggtggcact tttcggggaa atgtgcgcgg aacccctatt
     8941 tgtttatttt tctaaataca ttcaaatatg tatccgctca tgagacaata accctgataa
     9001 atgcttcaat aatattgaaa aaggaagagt atgagtattc aacatttccg tgtcgccctt
     9061 attccctttt ttgcggcatt ttgccttcct gtttttgctc acccagaaac gctggtgaaa
     9121 gtaaaagatg ctgaagatca gttgggtgca cgagtgggtt acatcgaact ggatctcaac
     9181 agcggtaaga tccttgagag ttttcgcccc gaagaacgtt ttccaatgat gagcactttt
     9241 aaagttctgc tatgtggcgc ggtattatcc cgtattgacg ccgggcaaga gcaactcggt
     9301 cgccgcatac actattctca gaatgacttg gttgagtact caccagtcac agaaaagcat
     9361 cttacggatg gcatgacagt aagagaatta tgcagtgctg ccataaccat gagtgataac
     9421 actgcggcca acttacttct gacaacgatc ggaggaccga aggagctaac cgcttttttg
     9481 cacaacatgg gggatcatgt aactcgcctt gatcgttggg aaccggagct gaatgaagcc
     9541 ataccaaacg acgagcgtga caccacgatg cctgtagcaa tggcaacaac gttgcgcaaa
     9601 ctattaactg gcgaactact tactctagct tcccggcaac aattaataga ctggatggag
     9661 gcggataaag ttgcaggacc acttctgcgc tcggcccttc cggctggctg gtttattgct
     9721 gataaatctg gagccggtga gcgtggaagc cgcggtatca ttgcagcact ggggccagat
     9781 ggtaagccct cccgtatcgt agttatctac acgacgggga gtcaggcaac tatggatgaa
     9841 cgaaatagac agatcgctga gataggtgcc tcactgatta agcattggta actgtcagac
     9901 caagtttact catatatact ttagattgat ttaaaacttc atttttaatt taaaaggatc
     9961 taggtgaaga tcctttttga taatctcatg accaaaatcc cttaacgtga gttttcgttc
    10021 cactgagcgt cagaccccgt agaaaagatc aaaggatctt cttgagatcc tttttttctg
    10081 cgcgtaatct gctgcttgca aacaaaaaaa ccaccgctac cagcggtggt ttgtttgccg
    10141 gatcaagagc taccaactct ttttccgaag gtaactggct tcagcagagc gcagatacca
    10201 aatactgttc ttctagtgta gccgtagtta ggccaccact tcaagaactc tgtagcaccg
    10261 cctacatacc tcgctctgct aatcctgtta ccagtggctg ctgccagtgg cgataagtcg
    10321 tgtcttaccg ggttggactc aagacgatag ttaccggata aggcgcagcg gtcgggctga
    10381 acggggggtt cgtgcacaca gcccagcttg gagcgaacga cctacaccga actgagatac
    10441 ctacagcgtg agctatgaga aagcgccacg cttcccgaag ggagaaaggc ggacaggtat
    10501 ccggtaagcg gcagggtcgg aacaggagag cgcacgaggg agcttccagg gggaaacgcc
    10561 tggtatcttt atagtcctgt cgggtttcgc cacctctgac ttgagcgtcg atttttgtga
    10621 tgctcgtcag gggggcggag cctatggaaa aacgccagca acgcggcctt tttacggttc
    10681 ctggcctttt gctggccttt tgctcacatg t
//
