LOCUS       sequence-220580-       10718 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 10718)
  TITLE     CRISPR-Cas9 system with SacB toxin Positive Selection
  JOURNAL   Unpublished
REFERENCE   2  (bases 1 to 10718)
  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            "Unpublished"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..10718
                     /mol_type="other DNA"
                     /organism="synthetic DNA construct"
     promoter        7..255
                     /label=U6 promoter
                     /note="RNA polymerase III promoter for human U6 snRNA 
                     (Domitrovich & Kunkel, 2003)"
     primer_bind     7..27
                     /label=hU6-F
                     /note="Human U6 promoter, forward primer"
     primer_bind     178..197
                     /label=LKO.1 5'
                     /note="Human U6 promoter, forward primer"
     CDS             join(279..365,366..1700)
                     /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        1709..1784
                     /label=gRNA scaffold
                     /note="guide RNA scaffold for the Streptococcus pyogenes 
                     CRISPR/Cas9 system"
     enhancer        1881..2166
                     /label=CMV enhancer
                     /note="human cytomegalovirus immediate early enhancer; 
                     contains an 18-bp deletion relative to the standard CMV 
                     enhancer"
     promoter        2168..2445
                     /label=chicken beta-actin promoter
     intron          2446..2674
                     /label=hybrid intron
                     /note="hybrid between chicken beta-actin (CBA) and minute 
                     virus of mice (MMV) introns (Gray et al., 2011)"
     regulatory      2686..2695
                     /label=Kozak sequence
                     /note="vertebrate consensus sequence for strong initiation 
                     of translation (Kozak, 1987)"
                     /regulatory_class="other"
     CDS             2695..2760
                     /codon_start=1
                     /product="three tandem FLAG(R) epitope tags, followed by an
                     enterokinase cleavage site"
                     /label=3xFLAG
                     /translation="DYKDHDGDYKDHDIDYKDDDDK"
     CDS             2767..2787
                     /codon_start=1
                     /product="nuclear localization signal of SV40 (simian virus
                     40) large T antigen"
                     /label=SV40 NLS
                     /translation="PKKKRKV"
     CDS             2812..6912
                     /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             2812..6912
                     /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             6913..6960
                     /codon_start=1
                     /product="bipartite nuclear localization signal from 
                     nucleoplasmin"
                     /label=nucleoplasmin NLS
                     /translation="KRPAATKKAGQAKKKK"
     CDS             6964..7017
                     /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             7024..7734
                     /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(7169..7187)
                     /label=mCherry-R
                     /note="mCherry, reverse primer"
     primer_bind     complement(7759..7776)
                     /label=BGH-rev
                     /note="Bovine growth hormone terminator, reverse primer. 
                     Also called BGH reverse"
     polyA_signal    7765..7972
                     /label=bGH poly(A) signal
                     /note="bovine growth hormone polyadenylation signal"
     repeat_region   7981..8121
                     /label=AAV2 ITR
                     /note="inverted terminal repeat of adeno-associated virus 
                     serotype 2"
     repeat_region   7981..8110
                     /label=AAV2 ITR
     rep_origin      8196..8651
                     /direction=RIGHT
                     /label=f1 ori
                     /note="f1 bacteriophage origin of replication; arrow 
                     indicates direction of (+) strand synthesis"
     primer_bind     complement(8283..8302)
                     /label=F1ori-R
                     /note="F1 origin, reverse primer"
     primer_bind     8493..8514
                     /label=F1ori-F
                     /note="F1 origin, forward primer"
     primer_bind     complement(8668..8687)
                     /label=pRS-marker
                     /note="pRS vectors, use to sequence yeast selectable 
                     marker"
     primer_bind     8787..8809
                     /label=pGEX 3'
                     /note="pGEX vectors, reverse primer"
     primer_bind     complement(8847..8865)
                     /label=pBRforEco
                     /note="pBR322 vectors, upsteam of EcoRI site, forward 
                     primer"
     promoter        8933..9037
                     /gene="bla"
                     /label=AmpR promoter
     CDS             join(9038..9106,9107..9898)
                     /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(9256..9275)
                     /label=Amp-R
                     /note="Ampicillin resistance gene, reverse primer"
     rep_origin      10069..10657
                     /direction=RIGHT
                     /label=ori
                     /note="high-copy-number ColE1/pMB1/pBR322/pUC origin of 
                     replication"
     primer_bind     10558..10577
                     /label=pBR322ori-F
                     /note="pBR322 origin, forward primer"
ORIGIN
        1 ccccacgagg gcctatttcc catgattcct tcatatttgc atatacgata caaggctgtt
       61 agagagataa ttggaattaa tttgactgta aacacaaaga tattagtaca aaatacgtga
      121 cgtagaaagt aataatttct tgggtagttt gcagttttaa aattatgttt taaaatggac
      181 tatcatatgc ttaccgtaac ttgaaagtat ttcgatttct tggctttata tatcttgtgg
      241 aaaggacgaa acaccgggtc ttcaaggaga catgaacgat gaacatcaaa aagtttgcaa
      301 aacaagcaac agtattaacc tttactaccg cactgctggc aggaggcgca actcaagcgt
      361 ttgcgaaaga aacgaaccaa aagccatata aggaaacata cggcatttcc catattacac
      421 gccatgatat gctgcaaatc cctgaacagc aaaaaaatga aaaatatcaa gttcctgaat
      481 tcgattcgtc cacaattaaa aatatctctt ctgcaaaagg cctggacgtt tgggacagct
      541 ggccattaca aaacgctgac ggcactgtcg caaactatca cggctaccac atcgtctttg
      601 cattagccgg agatcctaaa aatgcggatg acacatcgat ttacatgttc tatcaaaaag
      661 tcggcgaaac ttctattgac agctggaaaa acgctggccg cgtctttaaa gacagcgaca
      721 aattcgatgc aaatgattct atcctaaaag accaaacaca agaatggtca ggttcagcca
      781 catttacatc tgacggaaaa atccgtttat tctacactga tttctccggt aaacattacg
      841 gcaaacaaac actgacaact gcacaagtta acgtatcagc atcagacagc tctttgaaca
      901 tcaacggtgt agaggattat aaatcaatct ttgacggtga cggaaaaacg tatcaaaatg
      961 tacagcagtt catcgatgaa ggcaactaca gctcaggcga caaccatacg ctgagagatc
     1021 ctcactacgt agaagataaa ggccacaaat acttagtatt tgaagcaaac actggaactg
     1081 aagatggcta ccaaggcgaa gaatctttat ttaacaaagc atactatggc aaaagcacat
     1141 cattcttccg tcaagaaagt caaaaacttc tgcaaagcga taaaaaacgc acggctgagt
     1201 tagcaaacgg cgctctcggt atgattgagc taaacgatga ttacacactg aaaaaagtga
     1261 tgaaaccgct gattgcatct aacacagtaa cagatgaaat tgaacgcgcg aacgtcttta
     1321 aaatgaacgg caaatggtac ctgttcactg actcccgcgg atcaaaaatg acgattgacg
     1381 gcattacgtc taacgatatt tacatgcttg gttatgtttc taattcttta actggcccat
     1441 acaagccgct gaacaaaact ggccttgtgt taaaaatgga tcttgatcct aacgatgtaa
     1501 cctttactta ctcacacttc gctgtacctc aagcgaaagg aaacaatgtc gtgattacaa
     1561 gctatatgac aaacagagga ttctacgcag acaaacaatc aacgtttgcg ccaagcttcc
     1621 tgctgaacat caaaggcaag aaaacatctg ttgtcaaaga cagcatcctt gaacaaggac
     1681 aattaacagt taacaaataa gaagacctgt tttagagcta gaaatagcaa gttaaaataa
     1741 ggctagtccg ttatcaactt gaaaaagtgg caccgagtcg gtgctttttt gttttagagc
     1801 tagaaatagc aagttaaaat aaggctagtc cgtttttagc gcgtgcgcca attctgcaga
     1861 caaatggctc tagaggtacc cgttacataa cttacggtaa atggcccgcc tggctgaccg
     1921 cccaacgacc cccgcccatt gacgtcaata gtaacgccaa tagggacttt ccattgacgt
     1981 caatgggtgg agtatttacg gtaaactgcc cacttggcag tacatcaagt gtatcatatg
     2041 ccaagtacgc cccctattga cgtcaatgac ggtaaatggc ccgcctggca ttgtgcccag
     2101 tacatgacct tatgggactt tcctacttgg cagtacatct acgtattagt catcgctatt
     2161 accatggtcg aggtgagccc cacgttctgc ttcactctcc ccatctcccc cccctcccca
     2221 cccccaattt tgtatttatt tattttttaa ttattttgtg cagcgatggg ggcggggggg
     2281 gggggggggc gcgcgccagg cggggcgggg cggggcgagg ggcggggcgg ggcgaggcgg
     2341 agaggtgcgg cggcagccaa tcagagcggc gcgctccgaa agtttccttt tatggcgagg
     2401 cggcggcggc ggcggcccta taaaaagcga agcgcgcggc gggcgggagt cgctgcgacg
     2461 ctgccttcgc cccgtgcccc gctccgccgc cgcctcgcgc cgcccgcccc ggctctgact
     2521 gaccgcgtta ctcccacagg tgagcgggcg ggacggccct tctcctccgg gctgtaatta
     2581 gctgagcaag aggtaagggt ttaagggatg gttggttggt ggggtattaa tgtttaatta
     2641 cctggagcac ctgcctgaaa tcactttttt tcaggttgga ccggtgccac catggactat
     2701 aaggaccacg acggagacta caaggatcat gatattgatt acaaagacga tgacgataag
     2761 atggccccaa agaagaagcg gaaggtcggt atccacggag tcccagcagc cgacaagaag
     2821 tacagcatcg gcctggacat cggcaccaac tctgtgggct gggccgtgat caccgacgag
     2881 tacaaggtgc ccagcaagaa attcaaggtg ctgggcaaca ccgaccggca cagcatcaag
     2941 aagaacctga tcggagccct gctgttcgac agcggcgaaa cagccgaggc cacccggctg
     3001 aagagaaccg ccagaagaag atacaccaga cggaagaacc ggatctgcta tctgcaagag
     3061 atcttcagca acgagatggc caaggtggac gacagcttct tccacagact ggaagagtcc
     3121 ttcctggtgg aagaggataa gaagcacgag cggcacccca tcttcggcaa catcgtggac
     3181 gaggtggcct accacgagaa gtaccccacc atctaccacc tgagaaagaa actggtggac
     3241 agcaccgaca aggccgacct gcggctgatc tatctggccc tggcccacat gatcaagttc
     3301 cggggccact tcctgatcga gggcgacctg aaccccgaca acagcgacgt ggacaagctg
     3361 ttcatccagc tggtgcagac ctacaaccag ctgttcgagg aaaaccccat caacgccagc
     3421 ggcgtggacg ccaaggccat cctgtctgcc agactgagca agagcagacg gctggaaaat
     3481 ctgatcgccc agctgcccgg cgagaagaag aatggcctgt tcggaaacct gattgccctg
     3541 agcctgggcc tgacccccaa cttcaagagc aacttcgacc tggccgagga tgccaaactg
     3601 cagctgagca aggacaccta cgacgacgac ctggacaacc tgctggccca gatcggcgac
     3661 cagtacgccg acctgtttct ggccgccaag aacctgtccg acgccatcct gctgagcgac
     3721 atcctgagag tgaacaccga gatcaccaag gcccccctga gcgcctctat gatcaagaga
     3781 tacgacgagc accaccagga cctgaccctg ctgaaagctc tcgtgcggca gcagctgcct
     3841 gagaagtaca aagagatttt cttcgaccag agcaagaacg gctacgccgg ctacattgac
     3901 ggcggagcca gccaggaaga gttctacaag ttcatcaagc ccatcctgga aaagatggac
     3961 ggcaccgagg aactgctcgt gaagctgaac agagaggacc tgctgcggaa gcagcggacc
     4021 ttcgacaacg gcagcatccc ccaccagatc cacctgggag agctgcacgc cattctgcgg
     4081 cggcaggaag atttttaccc attcctgaag gacaaccggg aaaagatcga gaagatcctg
     4141 accttccgca tcccctacta cgtgggccct ctggccaggg gaaacagcag attcgcctgg
     4201 atgaccagaa agagcgagga aaccatcacc ccctggaact tcgaggaagt ggtggacaag
     4261 ggcgcttccg cccagagctt catcgagcgg atgaccaact tcgataagaa cctgcccaac
     4321 gagaaggtgc tgcccaagca cagcctgctg tacgagtact tcaccgtgta taacgagctg
     4381 accaaagtga aatacgtgac cgagggaatg agaaagcccg ccttcctgag cggcgagcag
     4441 aaaaaggcca tcgtggacct gctgttcaag accaaccgga aagtgaccgt gaagcagctg
     4501 aaagaggact acttcaagaa aatcgagtgc ttcgactccg tggaaatctc cggcgtggaa
     4561 gatcggttca acgcctccct gggcacatac cacgatctgc tgaaaattat caaggacaag
     4621 gacttcctgg acaatgagga aaacgaggac attctggaag atatcgtgct gaccctgaca
     4681 ctgtttgagg acagagagat gatcgaggaa cggctgaaaa cctatgccca cctgttcgac
     4741 gacaaagtga tgaagcagct gaagcggcgg agatacaccg gctggggcag gctgagccgg
     4801 aagctgatca acggcatccg ggacaagcag tccggcaaga caatcctgga tttcctgaag
     4861 tccgacggct tcgccaacag aaacttcatg cagctgatcc acgacgacag cctgaccttt
     4921 aaagaggaca tccagaaagc ccaggtgtcc ggccagggcg atagcctgca cgagcacatt
     4981 gccaatctgg ccggcagccc cgccattaag aagggcatcc tgcagacagt gaaggtggtg
     5041 gacgagctcg tgaaagtgat gggccggcac aagcccgaga acatcgtgat cgaaatggcc
     5101 agagagaacc agaccaccca gaagggacag aagaacagcc gcgagagaat gaagcggatc
     5161 gaagagggca tcaaagagct gggcagccag atcctgaaag aacaccccgt ggaaaacacc
     5221 cagctgcaga acgagaagct gtacctgtac tacctgcaga atgggcggga tatgtacgtg
     5281 gaccaggaac tggacatcaa ccggctgtcc gactacgatg tggaccatat cgtgcctcag
     5341 agctttctga aggacgactc catcgacaac aaggtgctga ccagaagcga caagaaccgg
     5401 ggcaagagcg acaacgtgcc ctccgaagag gtcgtgaaga agatgaagaa ctactggcgg
     5461 cagctgctga acgccaagct gattacccag agaaagttcg acaatctgac caaggccgag
     5521 agaggcggcc tgagcgaact ggataaggcc ggcttcatca agagacagct ggtggaaacc
     5581 cggcagatca caaagcacgt ggcacagatc ctggactccc ggatgaacac taagtacgac
     5641 gagaatgaca agctgatccg ggaagtgaaa gtgatcaccc tgaagtccaa gctggtgtcc
     5701 gatttccgga aggatttcca gttttacaaa gtgcgcgaga tcaacaacta ccaccacgcc
     5761 cacgacgcct acctgaacgc cgtcgtggga accgccctga tcaaaaagta ccctaagctg
     5821 gaaagcgagt tcgtgtacgg cgactacaag gtgtacgacg tgcggaagat gatcgccaag
     5881 agcgagcagg aaatcggcaa ggctaccgcc aagtacttct tctacagcaa catcatgaac
     5941 tttttcaaga ccgagattac cctggccaac ggcgagatcc ggaagcggcc tctgatcgag
     6001 acaaacggcg aaaccgggga gatcgtgtgg gataagggcc gggattttgc caccgtgcgg
     6061 aaagtgctga gcatgcccca agtgaatatc gtgaaaaaga ccgaggtgca gacaggcggc
     6121 ttcagcaaag agtctatcct gcccaagagg aacagcgata agctgatcgc cagaaagaag
     6181 gactgggacc ctaagaagta cggcggcttc gacagcccca ccgtggccta ttctgtgctg
     6241 gtggtggcca aagtggaaaa gggcaagtcc aagaaactga agagtgtgaa agagctgctg
     6301 gggatcacca tcatggaaag aagcagcttc gagaagaatc ccatcgactt tctggaagcc
     6361 aagggctaca aagaagtgaa aaaggacctg atcatcaagc tgcctaagta ctccctgttc
     6421 gagctggaaa acggccggaa gagaatgctg gcctctgccg gcgaactgca gaagggaaac
     6481 gaactggccc tgccctccaa atatgtgaac ttcctgtacc tggccagcca ctatgagaag
     6541 ctgaagggct cccccgagga taatgagcag aaacagctgt ttgtggaaca gcacaagcac
     6601 tacctggacg agatcatcga gcagatcagc gagttctcca agagagtgat cctggccgac
     6661 gctaatctgg acaaagtgct gtccgcctac aacaagcacc gggataagcc catcagagag
     6721 caggccgaga atatcatcca cctgtttacc ctgaccaatc tgggagcccc tgccgccttc
     6781 aagtactttg acaccaccat cgaccggaag aggtacacca gcaccaaaga ggtgctggac
     6841 gccaccctga tccaccagag catcaccggc ctgtacgaga cacggatcga cctgtctcag
     6901 ctgggaggcg acaaaaggcc ggcggccacg aaaaaggccg gccaggcaaa aaagaaaaag
     6961 cttgagggca gaggaagtct gctaacatgc ggtgacgtgg aggagaatcc cggccctgct
     7021 agcatggtga gcaagggcga ggaggataac atggccatca tcaaggagtt catgcgcttc
     7081 aaggtgcaca tggagggctc cgtgaacggc cacgagttcg agatcgaggg cgagggcgag
     7141 ggccgcccct acgagggcac ccagaccgcc aagctgaagg tgaccaaggg tggccccctg
     7201 cccttcgcct gggacatcct gtcccctcag ttcatgtacg gctccaaggc ctacgtgaag
     7261 caccccgccg acatccccga ctacttgaag ctgtccttcc ccgagggctt caagtgggag
     7321 cgcgtgatga acttcgagga cggcggcgtg gtgaccgtga cccaggactc ctccctgcag
     7381 gacggcgagt tcatctacaa ggtgaagctg cgcggcacca acttcccctc agacggcccc
     7441 gtaatgcaga agaaaaccat gggctgggag gcctcctccg agcggatgta ccccgaggac
     7501 ggcgccctga agggcgagat caagcagagg ctgaagctga aggacggcgg ccactacgac
     7561 gctgaggtca agaccaccta caaggccaag aagcccgtgc agctgcccgg cgcctacaac
     7621 gtcaacatca agttggacat cacctcccac aacgaggact acaccatcgt ggaacagtac
     7681 gaacgcgccg agggccgcca ctccaccggc ggcatggacg agctgtacaa gtaagaattc
     7741 ctagagctcg ctgatcagcc tcgactgtgc cttctagttg ccagccatct gttgtttgcc
     7801 cctcccccgt gccttccttg accctggaag gtgccactcc cactgtcctt tcctaataaa
     7861 atgaggaaat tgcatcgcat tgtctgagta ggtgtcattc tattctgggg ggtggggtgg
     7921 ggcaggacag caagggggag gattgggaag agaatagcag gcatgctggg gagcggccgc
     7981 aggaacccct agtgatggag ttggccactc cctctctgcg cgctcgctcg ctcactgagg
     8041 ccgggcgacc aaaggtcgcc cgacgcccgg gctttgcccg ggcggcctca gtgagcgagc
     8101 gagcgcgcag ctgcctgcag gggcgcctga tgcggtattt tctccttacg catctgtgcg
     8161 gtatttcaca ccgcatacgt caaagcaacc atagtacgcg ccctgtagcg gcgcattaag
     8221 cgcggcgggt gtggtggtta cgcgcagcgt gaccgctaca cttgccagcg ccctagcgcc
     8281 cgctcctttc gctttcttcc cttcctttct cgccacgttc gccggctttc cccgtcaagc
     8341 tctaaatcgg gggctccctt tagggttccg atttagtgct ttacggcacc tcgaccccaa
     8401 aaaacttgat ttgggtgatg gttcacgtag tgggccatcg ccctgataga cggtttttcg
     8461 ccctttgacg ttggagtcca cgttctttaa tagtggactc ttgttccaaa ctggaacaac
     8521 actcaaccct atctcgggct attcttttga tttataaggg attttgccga tttcggccta
     8581 ttggttaaaa aatgagctga tttaacaaaa atttaacgcg aattttaaca aaatattaac
     8641 gtttacaatt ttatggtgca ctctcagtac aatctgctct gatgccgcat agttaagcca
     8701 gccccgacac ccgccaacac ccgctgacgc gccctgacgg gcttgtctgc tcccggcatc
     8761 cgcttacaga caagctgtga ccgtctccgg gagctgcatg tgtcagaggt tttcaccgtc
     8821 atcaccgaaa cgcgcgagac gaaagggcct cgtgatacgc ctatttttat aggttaatgt
     8881 catgataata atggtttctt agacgtcagg tggcactttt cggggaaatg tgcgcggaac
     8941 ccctatttgt ttatttttct aaatacattc aaatatgtat ccgctcatga gacaataacc
     9001 ctgataaatg cttcaataat attgaaaaag gaagagtatg agtattcaac atttccgtgt
     9061 cgcccttatt cccttttttg cggcattttg ccttcctgtt tttgctcacc cagaaacgct
     9121 ggtgaaagta aaagatgctg aagatcagtt gggtgcacga gtgggttaca tcgaactgga
     9181 tctcaacagc ggtaagatcc ttgagagttt tcgccccgaa gaacgttttc caatgatgag
     9241 cacttttaaa gttctgctat gtggcgcggt attatcccgt attgacgccg ggcaagagca
     9301 actcggtcgc cgcatacact attctcagaa tgacttggtt gagtactcac cagtcacaga
     9361 aaagcatctt acggatggca tgacagtaag agaattatgc agtgctgcca taaccatgag
     9421 tgataacact gcggccaact tacttctgac aacgatcgga ggaccgaagg agctaaccgc
     9481 ttttttgcac aacatggggg atcatgtaac tcgccttgat cgttgggaac cggagctgaa
     9541 tgaagccata ccaaacgacg agcgtgacac cacgatgcct gtagcaatgg caacaacgtt
     9601 gcgcaaacta ttaactggcg aactacttac tctagcttcc cggcaacaat taatagactg
     9661 gatggaggcg gataaagttg caggaccact tctgcgctcg gcccttccgg ctggctggtt
     9721 tattgctgat aaatctggag ccggtgagcg tggaagccgc ggtatcattg cagcactggg
     9781 gccagatggt aagccctccc gtatcgtagt tatctacacg acggggagtc aggcaactat
     9841 ggatgaacga aatagacaga tcgctgagat aggtgcctca ctgattaagc attggtaact
     9901 gtcagaccaa gtttactcat atatacttta gattgattta aaacttcatt tttaatttaa
     9961 aaggatctag gtgaagatcc tttttgataa tctcatgacc aaaatccctt aacgtgagtt
    10021 ttcgttccac tgagcgtcag accccgtaga aaagatcaaa ggatcttctt gagatccttt
    10081 ttttctgcgc gtaatctgct gcttgcaaac aaaaaaacca ccgctaccag cggtggtttg
    10141 tttgccggat caagagctac caactctttt tccgaaggta actggcttca gcagagcgca
    10201 gataccaaat actgtccttc tagtgtagcc gtagttaggc caccacttca agaactctgt
    10261 agcaccgcct acatacctcg ctctgctaat cctgttacca gtggctgctg ccagtggcga
    10321 taagtcgtgt cttaccgggt tggactcaag acgatagtta ccggataagg cgcagcggtc
    10381 gggctgaacg gggggttcgt gcacacagcc cagcttggag cgaacgacct acaccgaact
    10441 gagataccta cagcgtgagc tatgagaaag cgccacgctt cccgaaggga gaaaggcgga
    10501 caggtatccg gtaagcggca gggtcggaac aggagagcgc acgagggagc ttccaggggg
    10561 aaacgcctgg tatctttata gtcctgtcgg gtttcgccac ctctgacttg agcgtcgatt
    10621 tttgtgatgc tcgtcagggg ggcggagcct atggaaaaac gccagcaacg cggccttttt
    10681 acggttcctg gccttttgct ggccttttgc tcacatgt
//
