LOCUS       sequence-407164-       10292 bp    DNA     circular SYN 14-APR-2025
DEFINITION  3XHA tagged dead en1Cas9 from F. novicida with KRAB repressor 
            domain, T2A self-cleaving peptide and enhanced GFP biomarker for 
            mammalian expression.
ACCESSION   .
VERSION     .
KEYWORDS    .
SOURCE      synthetic DNA construct
  ORGANISM  synthetic DNA construct
REFERENCE   1  (bases 1 to 10292)
  TITLE     3XHA tagged dead en1Cas9 from F. novicida with KRAB repressor 
            domain, T2A self-cleaving peptide region and enhanced GFP biomarker 
            for mammalian expression
  JOURNAL   Unpublished
REFERENCE   2  (bases 1 to 10292)
  AUTHORS   .
  TITLE     Direct Submission
  JOURNAL   Exported Apr 14, 2025 from SnapGene Server 8.0.1
            https://www.snapgene.com
COMMENT     SGRef: number: 1; type: "Journal Article"; journalName: 
            "Unpublished"
COMMENT     Sequence Label: PX458_3XHA_den1FnCas9_KRAB_T2A_eGFP
FEATURES             Location/Qualifiers
     source          1..10292
                     /mol_type="other DNA"
                     /organism="synthetic DNA construct"
     promoter        24..272
                     /label=U6 promoter
                     /note="RNA polymerase III promoter for human U6 snRNA 
                     (Domitrovich & Kunkel, 2003)"
     primer_bind     24..44
                     /label=hU6-F
                     /note="Human U6 promoter, forward primer"
     primer_bind     195..214
                     /label=LKO.1 5'
                     /note="Human U6 promoter, forward primer"
     misc_RNA        291..363
                     /label=Fn gRNA scaffold
                     /note="guide RNA scaffold for the Francisella novicida 
                     CRISPR/Cas9 system (Hirano et al., 2016)"
     enhancer        460..745
                     /label=CMV enhancer
                     /note="human cytomegalovirus immediate early enhancer; 
                     contains an 18-bp deletion relative to the standard CMV 
                     enhancer"
     intron          997..1224
                     /label=hybrid intron
                     /note="hybrid between chicken beta-actin (CBA) and minute 
                     virus of mice (MMV) introns (Gray et al., 2011)"
     regulatory      1236..1245
                     /label=Kozak sequence
                     /note="vertebrate consensus sequence for strong initiation 
                     of translation (Kozak, 1987)"
                     /regulatory_class="other"
     CDS             1251..1331
                     /codon_start=1
                     /product="three tandem HA epitope tags"
                     /label=3xHA
                     /translation="YPYDVPDYAYPYDVPDYAYPYDVPDYA"
     CDS             1338..1358
                     /codon_start=1
                     /product="nuclear localization signal of SV40 (simian virus
                     40) large T antigen"
                     /label=SV40 NLS
                     /translation="PKKKRKV"
     CDS             1383..6266
                     /codon_start=1
                     /product="Cas9 endonuclease from the Francisella novicida 
                     Type II CRISPR/Cas system (Hirano et al., 2016)"
                     /label=FnCas9
                     /translation="NFKILPIAIALGVKNTGVFSAFYQKGTSLERLDNKNGKVYELSKD
                     SYTLLMNNRTARRHQRRGIDRKQLVKRLFKLIWTEQLNLEWDKDTQQAISFLFNRRGFS
                     FITDGYSPEYLNIVPEQVKAILMDIFDDYNGEDDLDSYLKLATEQESKISEIYNKLMQK
                     ILEFKLMKLCTDIKDDKVSTKTLKEITSYEFELLADYLANYSESLKTQKFSYTDKQGNL
                     KELSYYHHDKYNIQEFLKRHATINDRILDTLLTDDLDIWNFNFEKFDFDKNEEKLQNQE
                     DKDHIQAHLHHFVFAVNKIKSEMASGGRHRSQYFQEITNVLDENNHQEGYLKNFCENLH
                     NKKYSNLSVKNLVNLIGNLSNLELKPLRKYFNDKIHAKADHWDEQKFTETYCHWILGEW
                     RVGVKDQDKKDGAKYSYKDLCNELKQKVTKAGLVDFLLELDPCRTIPPYLDNNNRKPPK
                     CQSLILNPKFLDNQYPNWQQYLQELKKLQSIQNYLDSFETDLKVLKSSKDQPYFVEYKS
                     SNQQIASGQRDYKDLDARILQFIFDRVKASDELLLNEIYFQAKKLKQKASSELEKLESS
                     KKLDEVIANSQLSQILKSQHTNGIFEQGTFLHLVCKYYKQRQRARDSRLYIMPEYRYDK
                     KLHKYNNTGRFDDDNQLLTYCNHKPRQKRYQLLNDLAGVLQVSPNFLKDKIGSDDDLFI
                     SKWLVEHIRGFKKACEDSLKIQKDNRGLLNHKINIARNTKGKCEKEIFNLICKIEGSED
                     KKGNYKHGLAYELGVLLFGEPNEASKPEFDRKIKKFNSIYSFAQIQQIAFAERKGNANT
                     CAVCSADNAHRMQQIKITEPVEDNKDKIILSAKAQRLPAIPTRIVDGAVKKMATILAKN
                     IVDDNWQNIKQVLSAKHQLHIPIITESNAFEFEPALADVKGKSLKDRRKKALERISPEN
                     IFKDKNNRIKEFAKGISAYSGANLTDGDFDGAKEELDAIIPRSHKKYGTLNDEANLICV
                     TRGDNKNKGNRIFCLRDLADNYKLKQFETTDDLEIEKKIADTIWDANKKDFKFGNYRSF
                     INLTPQEQKAFRHALFLADENPIKQAVIRAINNRNRTFVNGTQRYFAEVLANNIYLRAK
                     KENLNTDKISFDYFGIPTIGNGRGIAEIRQLYEKVDSDIQAYAKGDKPQASYSHLIDAM
                     LAFCIAADEHRNDGSIGLEIDKNYSLYPLDKNTGEVFTKDIFSQIKITDNEFSDKKLVR
                     KKAIEGFNTHRQMTRDGIYAENYLPILIHKELNEVRKGYTWKNSEEIKIFKGKKYDIQQ
                     LNNLVYCLKFVDKPISIDIQISTLEELRNILTTNNIAATAEYYYINLKTQKLHEYYIEN
                     YNTALGYKKYSKEMEFLRSLAYRSRRVKIKSIDDVKQVLDKDSNFIIGKITLPFKKEWQ
                     RLYREWQNTTIKDDYEFLKSFFNVKSITKLHKKVRKDFSLPISTNEGKFLVKRKTWDNN
                     FIYQILNDSDSRADGTKPFIPAFDISKNEIVEAIIDSFTSKNIFWLPKNIELQKVDNKN
                     IFAIDTSKWFEVETPSDLRDIGIATIQYKIDNNSRPKVRVKLDYVIDDDSKINYFMNHS
                     LLKSRYPDKVLEILKQSTIIEFESSGFNKTIKEMLGMKLAGIYNETSNN"
     CDS             6267..6314
                     /codon_start=1
                     /product="bipartite nuclear localization signal from 
                     nucleoplasmin"
                     /label=nucleoplasmin NLS
                     /translation="KRPAATKKAGQAKKKK"
     CDS             6360..6545
                     /codon_start=1
                     /product="Kruppel-associated box (KRAB) transcriptional 
                     repression domain from the human zinc finger protein ZNF10 
                     (Margolin et al., 1994)"
                     /label=KRAB
                     /translation="RTLVTFKDVFVDFTREEWKLLDTAQQIVYRNVMLENYKNLVSLGY
                     QLTKPDVILRLEKGEEP"
     CDS             6561..6614
                     /codon_start=1
                     /product="2A peptide from Thosea asigna virus capsid 
                     protein"
                     /label=T2A
                     /note="Eukaryotic ribosomes fail to insert a peptide bond 
                     between the Gly and Pro residues, yielding separate 
                     polypeptides."
                     /translation="EGRGSLLTCGDVEENPGP"
     CDS             join(6615..6617,6618..7328)
                     /codon_start=1
                     /product="enhanced GFP"
                     /label=EGFP
                     /translation="VSKGEELFTGVVPILVELDGDVNGHKFSVSGEGEGDATYGKLTLK
                     FICTTGKLPVPWPTLVTTLTYGVQCFSRYPDHMKQHDFFKSAMPEGYVQERTIFFKDDG
                     NYKTRAEVKFEGDTLVNRIELKGIDFKEDGNILGHKLEYNYNSHNVYIMADKQKNGIKV
                     NFKIRHNIEDGSVQLADHYQQNTPIGDGPVLLPDNHYLSTQSALSKDPNEKRDHMVLLE
                     FVTAAGITLGMDELYK"
     CDS             join(6615..6617,6618..7328)
                     /codon_start=1
                     /product="the original enhanced GFP (Yang et al., 1996)"
                     /label=EGFP
                     /note="mammalian codon-optimized"
                     /translation="VSKGEELFTGVVPILVELDGDVNGHKFSVSGEGEGDATYGKLTLK
                     FICTTGKLPVPWPTLVTTLTYGVQCFSRYPDHMKQHDFFKSAMPEGYVQERTIFFKDDG
                     NYKTRAEVKFEGDTLVNRIELKGIDFKEDGNILGHKLEYNYNSHNVYIMADKQKNGIKV
                     NFKIRHNIEDGSVQLADHYQQNTPIGDGPVLLPDNHYLSTQSALSKDPNEKRDHMVLLE
                     FVTAAGITLGMDELYK"
     primer_bind     complement(6657..6678)
                     /label=EGFP-N
                     /note="EGFP, reverse primer"
     primer_bind     complement(6918..6937)
                     /label=EXFP-R
                     /note="For distinguishing EGFP variants, reverse primer"
     primer_bind     7265..7286
                     /label=EGFP-C
                     /note="EGFP, forward primer"
     primer_bind     complement(7356..7373)
                     /label=BGH-rev
                     /note="Bovine growth hormone terminator, reverse primer. 
                     Also called BGH reverse"
     polyA_signal    7362..7569
                     /label=bGH poly(A) signal
                     /note="bovine growth hormone polyadenylation signal"
     repeat_region   7578..7718
                     /label=AAV2 ITR
                     /note="inverted terminal repeat of adeno-associated virus 
                     serotype 2"
     repeat_region   7578..7707
                     /label=AAV2 ITR
     rep_origin      7793..8248
                     /direction=RIGHT
                     /label=f1 ori
                     /note="f1 bacteriophage origin of replication; arrow 
                     indicates direction of (+) strand synthesis"
     primer_bind     complement(7880..7899)
                     /label=F1ori-R
                     /note="F1 origin, reverse primer"
     primer_bind     8090..8111
                     /label=F1ori-F
                     /note="F1 origin, forward primer"
     primer_bind     complement(8265..8284)
                     /label=pRS-marker
                     /note="pRS vectors, use to sequence yeast selectable 
                     marker"
     primer_bind     8384..8406
                     /label=pGEX 3'
                     /note="pGEX vectors, reverse primer"
     primer_bind     complement(8444..8462)
                     /label=pBRforEco
                     /note="pBR322 vectors, upsteam of EcoRI site, forward 
                     primer"
     promoter        8530..8634
                     /gene="bla"
                     /label=AmpR promoter
     CDS             join(8635..8703,8704..9495)
                     /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(8853..8872)
                     /label=Amp-R
                     /note="Ampicillin resistance gene, reverse primer"
     rep_origin      9666..10254
                     /direction=RIGHT
                     /label=ori
                     /note="high-copy-number ColE1/pMB1/pBR322/pUC origin of 
                     replication"
     primer_bind     10155..10174
                     /label=pBR322ori-F
                     /note="pBR322 origin, forward primer"
ORIGIN
        1 ttgctggcct tttgctcaca tgtgagggcc tatttcccat gattccttca tatttgcata
       61 tacgatacaa ggctgttaga gagataattg gaattaattt gactgtaaac acaaagatat
      121 tagtacaaaa tacgtgacgt agaaagtaat aatttcttgg gtagtttgca gttttaaaat
      181 tatgttttaa aatggactat catatgctta ccgtaacttg aaagtatttc gatttcttgg
      241 ctttatatat cttgtggaaa ggacgaaaca ccgggtcttc gagaagacct gtttcagttg
      301 cgccgaaagg cgctctgtaa tcatttaaaa gtattttgaa cggacctctg tttgacacgt
      361 ctgttttttg ttttagagct agaaatagca agttaaaata aggctagtcc gtttttagcg
      421 cgtgcgccaa ttctgcagac aaatggctct agaggtaccc gttacataac ttacggtaaa
      481 tggcccgcct ggctgaccgc ccaacgaccc ccgcccattg acgtcaatag taacgccaat
      541 agggactttc cattgacgtc aatgggtgga gtatttacgg taaactgccc acttggcagt
      601 acatcaagtg tatcatatgc caagtacgcc ccctattgac gtcaatgacg gtaaatggcc
      661 cgcctggcat tgtgcccagt acatgacctt atgggacttt cctacttggc agtacatcta
      721 cgtattagtc atcgctatta ccatggtcga ggtgagcccc acgttctgct tcactctccc
      781 catctccccc ccctccccac ccccaatttt gtatttattt attttttaat tattttgtgc
      841 agcgatgggg gcggggcggg gcggggcgag gggcggggcg gggcgaggcg gagaggtgcg
      901 gcggcagcca atcagagcgg cgcgctccga aagtttcctt ttatggcgag gcggcggcgg
      961 cggcggccct ataaaaagcg aagcgcgcgg cgggcgggag tcgctgcgcg ctgccttcgc
     1021 cccgtgcccc gctccgccgc cgcctcgcgc cgcccgcccc ggctctgact gaccgcgtta
     1081 ctcccacagg tgagcgggcg ggacggccct tctcctccgg gctgtaatta gctgagcaag
     1141 aggtaagggt ttaagggatg gttggttggt ggggtattaa tgtttaatta cctggagcac
     1201 ctgcctgaaa tcactttttt tcaggttgga ccggtgccac catgggatcc tacccatacg
     1261 atgttccaga ttacgcttac ccatacgatg ttccagatta cgcttaccca tacgatgttc
     1321 cagattacgc tatggcccca aagaagaagc ggaaggtcag tatccacgga gtcccagcag
     1381 ccaattttaa gatcctgcct atcgcaatcg ccctgggcgt caagaatact ggcgtgttta
     1441 gtgcttttta tcagaagggg acctcactgg agagactgga caataagaac ggaaaagtgt
     1501 atgaactgtc caaggattct tacactctgc tgatgaacaa taggaccgca cggagacacc
     1561 agaggcgagg aattgacagg aaacagctgg tgaagcgcct gttcaaactg atctggacag
     1621 agcagctgaa cctggaatgg gataaggaca ctcagcaggc catcagcttc ctgtttaatc
     1681 gacggggatt ctcttttatt actgacggct atagtcctga gtacctgaac atcgtgccag
     1741 aacaggtcaa ggcaatcctg atggacattt tcgacgatta taatggcgag gacgatctgg
     1801 attcctacct gaaactggcc acagagcaag agagtaagat cagcgaaatc tacaacaagc
     1861 tgatgcagaa gatcctggag ttcaagctga tgaaactgtg caccgacatc aaggacgata
     1921 aagtgagtac caagacactg aaagagatca caagctacga gttcgaactg ctggccgatt
     1981 atctggctaa ctacagcgaa tccctgaaaa cccagaaatt ttcctacaca gacaagcagg
     2041 gcaatctgaa agagctgtct tactaccacc atgataagta caacatccag gagttcctga
     2101 agagacacgc caccatcaat gacaggattc tggatacact gctgactgac gatctggaca
     2161 tctggaactt caacttcgag aagttcgatt tcgacaagaa cgaggaaaaa ctgcagaatc
     2221 aggaagataa ggaccacatt caggctcatc tgcaccattt cgtgtttgca gtcaataaga
     2281 tcaaaagcga gatggcatcc ggcgggcgcc atcgaagcca gtacttccag gaaatcacca
     2341 acgtgctgga cgagaacaat caccaggaag gctacctgaa aaacttctgt gagaatctgc
     2401 ataacaagaa gtacagcaat ctgtccgtga agaatctggt caacctgatt ggaaatctgt
     2461 ccaacctgga actgaagccc ctgcgcaaat acttcaacga caagatccac gctaaagcag
     2521 accattggga tgagcagaag tttactgaaa cctattgcca ctggattctg ggcgagtggc
     2581 gggtgggggt caaggatcag gacaagaaag acggcgcaaa gtattcttac aaggacctgt
     2641 gtaacgagct gaagcagaaa gtgactaagg ccgggctggt ggacttcctg ctggagctgg
     2701 acccctgccg aaccattcca ccttacctgg acaacaataa cagaaagcca cccaaatgtc
     2761 agagcctgat cctgaatccc aagtttctgg ataatcagta tcctaactgg cagcagtacc
     2821 tgcaggagct gaagaaactg cagtcaatcc agaactacct ggacagcttc gaaaccgatc
     2881 tgaaggtgct gaaaagctcc aaggaccagc cttacttcgt cgagtacaag tctagtaacc
     2941 agcagatcgc ttccggccag cgggattaca aggatctgga cgcaagaatc ctgcagttca
     3001 tttttgacag ggtgaaggcc tctgatgagc tgctgctgaa cgaaatctat ttccaggcaa
     3061 agaaactgaa gcagaaagcc tcaagcgagc tggaaaagct ggagtcctct aagaaactgg
     3121 acgaagtgat cgctaactct cagctgagtc agattctgaa gtctcagcac acaaatggaa
     3181 tcttcgagca gggcactttt ctgcatctgg tgtgcaaata ctataagcag cgacagagag
     3241 ccagggacag ccgcctgtac atcatgcctg aatatcgata cgataagaaa ctgcacaagt
     3301 acaacaacac cggccgcttt gacgatgaca accagctgct gacatattgt aatcataagc
     3361 cccggcagaa aagataccag ctgctgaacg acctggcagg agtgctgcag gtctctccta
     3421 attttctgaa ggataaaatc gggtccgatg acgatctgtt catttctaag tggctggtgg
     3481 agcacatccg gggctttaag aaggcctgcg aagatagcct gaaaatccag aaggataaca
     3541 ggggactgct gaatcataag atcaacattg cacgcaatac caagggcaaa tgcgagaaag
     3601 aaatcttcaa cctgatctgt aagattgagg ggagcgaaga taagaaaggg aattataagc
     3661 acggactggc ctacgagctg ggagtgctgc tgttcggaga gccaaacgag gccagcaagc
     3721 ccgaatttga taggaaaatc aagaaattca attcaatcta cagctttgcc cagatccagc
     3781 agattgcctt tgctgagagg aaggggaatg caaacacatg cgccgtgtgt agtgcagaca
     3841 acgcccatcg catgcagcag atcaaaatta ctgagccagt cgaagataat aaggataaaa
     3901 tcattctgtc agcaaaggca cagcgactgc ctgcaatccc aacccgaatt gtggatggag
     3961 ctgtcaagaa aatggctaca attctggcaa agaatatcgt ggacgataat tggcagaaca
     4021 ttaagcaggt cctgagcgca aaacaccagc tgcatatccc aatcattacc gagtccaacg
     4081 ccttcgagtt tgaacccgct ctggcagacg tgaagggcaa atctctgaag gatagaagga
     4141 agaaagccct ggagcgaatt agtcccgaaa acatcttcaa ggataagaac aacagaatca
     4201 aggagtttgc taaggggatt tccgcctact ctggagctaa cctgacagat ggggacttcg
     4261 atggagcaaa ggaggaactg gatgccatca ttcctcgcag ccataagaaa tatggcactc
     4321 tgaacgacga ggctaatctg atttgcgtga cccggggcga taataagaac aaagggaacc
     4381 ggatcttctg tctgagagac ctggccgata attacaagct gaaacagttt gagaccacag
     4441 acgatctgga gatcgaaaag aaaattgccg acaccatctg ggatgctaat aagaaggact
     4501 tcaagttcgg aaactatcgg agcttcatca atctgacacc tcaggagcag aaagcattca
     4561 gacacgccct gtttctggct gatgaaaacc caatcaagca ggcagtgatc agagccatta
     4621 ataaccgcaa ccgaaccttc gtgaatggca cacagaggta ttttgctgag gtcctggcaa
     4681 ataacatcta cctgcgcgcc aagaaagaaa atctgaacac tgacaagatc agcttcgatt
     4741 actttggaat ccctaccatt ggaaacggcc gagggatcgc tgagattcgg cagctgtatg
     4801 aaaaggtgga cagtgatatc caggcctacg ctaaaggcga caagccacag gcctcttata
     4861 gtcacctgat tgatgctatg ctggcattct gcatcgccgc tgacgagcat cggaacgatg
     4921 gatctattgg cctggaaatc gacaaaaact atagtctgta ccctctggat aagaatactg
     4981 gcgaggtgtt caccaaagac atcttttcac agatcaagat taccgacaac gagttcagcg
     5041 ataagaaact ggtcagaaag aaagctattg aagggtttaa cacacacaga cagatgacta
     5101 gggatggaat ctatgcagag aattacctgc ctatcctgat tcataaggag ctgaacgaag
     5161 tgaggaaggg gtacacatgg aaaaattccg aggaaatcaa aattttcaag ggaaagaaat
     5221 acgacatcca gcagctgaat aacctggtgt attgtctgaa gtttgtggac aaaccaatca
     5281 gtattgatat ccagatttca accctggagg aactgagaaa catcctgact accaataaca
     5341 ttgcagccac tgccgagtac tattacatta atctgaaaac ccagaagctg cacgagtatt
     5401 acatcgaaaa ttacaacaca gccctggggt ataagaaata cagcaaggag atggagttcc
     5461 tgaggtccct ggcttatagg tctaggcgcg tgaagatcaa aagtattgac gatgtcaagc
     5521 aggtcctgga caaggattca aacttcatca tcggaaagat cacactgccc ttcaagaaag
     5581 agtggcagcg actgtaccgg gaatggcaga acacaactat caaagacgat tatgagtttc
     5641 tgaagagctt ctttaatgtg aagtccatta ctaaactgca caagaaagtc cggaaagact
     5701 tctctctgcc catcagtaca aacgagggca agtttctggt gaagagaaaa acttgggata
     5761 ataacttcat ctaccagatt ctgaatgact cagatagcag ggcagacggg actaaaccct
     5821 tcattcctgc ctttgatatc agcaagaacg agattgtgga agccatcatt gacagtttca
     5881 cctcaaaaaa catcttttgg ctgccaaaga atattgagct gcagaaggtg gacaacaaga
     5941 acatcttcgc cattgatacc agcaagtggt ttgaggtcga aacaccatcc gacctgcgcg
     6001 atatcggcat tgctaccatt cagtacaaga tcgacaataa ctcacgcccc aaggtgcgag
     6061 tcaaactgga ttacgtgatc gacgatgaca gcaagattaa ctatttcatg aatcactcac
     6121 tgctgaagag ccggtatccc gacaaagtcc tggagatcct gaagcagagc acaatcattg
     6181 agttcgaaag ttcagggttt aacaaaacta ttaaggagat gctgggaatg aagctggccg
     6241 gcatctacaa tgaaacctcc aataacaaaa ggccggcggc cacgaaaaag gccggccagg
     6301 caaaaaagaa aaagggcggt ggcggaggga tggatgctaa gtcactaact gcctggtccc
     6361 ggacactggt gaccttcaag gatgtatttg tggacttcac cagggaggag tggaagctgc
     6421 tggacactgc tcagcagatc gtgtacagaa atgtgatgct ggagaactat aagaacctgg
     6481 tttccttggg ttatcagctt actaagccag atgtgatcct ccggttggag aagggagaag
     6541 agcccgaatt cggcagtgga gagggcagag gaagtctgct aacatgcggt gacgtcgagg
     6601 agaatcctgg cccagtgagc aagggcgagg agctgttcac cggggtggtg cccatcctgg
     6661 tcgagctgga cggcgacgta aacggccaca agttcagcgt gtccggcgag ggcgagggcg
     6721 atgccaccta cggcaagctg accctgaagt tcatctgcac caccggcaag ctgcccgtgc
     6781 cctggcccac cctcgtgacc accctgacct acggcgtgca gtgcttcagc cgctaccccg
     6841 accacatgaa gcagcacgac ttcttcaagt ccgccatgcc cgaaggctac gtccaggagc
     6901 gcaccatctt cttcaaggac gacggcaact acaagacccg cgccgaggtg aagttcgagg
     6961 gcgacaccct ggtgaaccgc atcgagctga agggcatcga cttcaaggag gacggcaaca
     7021 tcctggggca caagctggag tacaactaca acagccacaa cgtctatatc atggccgaca
     7081 agcagaagaa cggcatcaag gtgaacttca agatccgcca caacatcgag gacggcagcg
     7141 tgcagctcgc cgaccactac cagcagaaca cccccatcgg cgacggcccc gtgctgctgc
     7201 ccgacaacca ctacctgagc acccagtccg ccctgagcaa agaccccaac gagaagcgcg
     7261 atcacatggt cctgctggag ttcgtgaccg ccgccgggat cactctcggc atggacgagc
     7321 tgtacaagga gttctaacta gagctcgctg atcagcctcg actgtgcctt ctagttgcca
     7381 gccatctgtt gtttgcccct cccccgtgcc ttccttgacc ctggaaggtg ccactcccac
     7441 tgtcctttcc taataaaatg aggaaattgc atcgcattgt ctgagtaggt gtcattctat
     7501 tctggggggt ggggtggggc aggacagcaa gggggaggat tgggaagaga atagcaggca
     7561 tgctggggag cggccgcagg aacccctagt gatggagttg gccactccct ctctgcgcgc
     7621 tcgctcgctc actgaggccg ggcgaccaaa ggtcgcccga cgcccgggct ttgcccgggc
     7681 ggcctcagtg agcgagcgag cgcgcagctg cctgcagggg cgcctgatgc ggtattttct
     7741 ccttacgcat ctgtgcggta tttcacaccg catacgtcaa agcaaccata gtacgcgccc
     7801 tgtagcggcg cattaagcgc ggcgggtgtg gtggttacgc gcagcgtgac cgctacactt
     7861 gccagcgcct tagcgcccgc tcctttcgct ttcttccctt cctttctcgc cacgttcgcc
     7921 ggctttcccc gtcaagctct aaatcggggg ctccctttag ggttccgatt tagtgcttta
     7981 cggcacctcg accccaaaaa acttgatttg ggtgatggtt cacgtagtgg gccatcgccc
     8041 tgatagacgg tttttcgccc tttgacgttg gagtccacgt tctttaatag tggactcttg
     8101 ttccaaactg gaacaacact caactctatc tcgggctatt cttttgattt ataagggatt
     8161 ttgccgattt cggtctattg gtttaaaaat gagctgattt aacaaaaatt taacgcgaat
     8221 tttaacaaaa tattaacgtt tacaatttta tggtgcactc tcagtacaat ctgctctgat
     8281 gccgcatagt taagccagcc ccgacacccg ccaacacccg ctgacgcgcc ctgacgggct
     8341 tgtctgctcc cagcatccgc ttacagacaa gctgtgaccg tctccgggag ctgcatgtgt
     8401 cagaggtttt caccgtcatc accgaaacgc gcgagacgaa agggcctcgt gatacgccta
     8461 tttttatagg ttaatgtcat gataataatg gtttcttaga cgtcaggtgg cacttttcgg
     8521 ggaaatgtgc gcggaacccc tatttgttta tttttctaaa tacattcaaa tatgtatccg
     8581 ctcatgagac aataaccctg ataaatgctt caataatatt gaaaaaggaa gagtatgagt
     8641 attcaacatt tccgtgtcgc ccttattccc ttttttgcgg cattttgcct tcctgttttt
     8701 gctcacccag aaacgctggt gaaagtaaaa gatgctgaag atcagttggg tgcacgagtg
     8761 ggttacatcg aactggatct caacagcggt aagatccttg agagttttcg ccccgaagaa
     8821 cgttttccaa tgatgagcac ttttaaagtt ctgctatgtg gcgcggtatt atcccgtatt
     8881 gacgccgggc aagagcaact cggtcgccgc atacactatt ctcagaatga cttggttgag
     8941 tactcaccag tcacagaaaa gcatcttacg gatggcatga cagtaagaga attatgcagt
     9001 gctgccataa ccatgagtga taacactgcg gccaacttac ttctgacaac gatcggagga
     9061 ccgaaggagc taaccgcttt tttgcacaac atgggggatc atgtaactcg ccttgatcgt
     9121 tgggaaccgg agctgaatga agccatacca aacgacgagc gtgacaccac gatgcctgta
     9181 gcaatggcaa caacgttgcg caaactatta actggcgaac tacttactct agcttcccgg
     9241 caacaattaa tagactggat ggaggcggat aaagttgcag gaccacttct gcgctcggcc
     9301 cttccggctg gctggtttat tgctgataaa tctggagccg gtgagcgtgg aagccgcggt
     9361 atcattgcag cactggggcc agatggtaag ccctcccgta tcgtagttat ctacacgacg
     9421 gggagtcagg caactatgga tgaacgaaat agacagatcg ctgagatagg tgcctcactg
     9481 attaagcatt ggtaactgtc agaccaagtt tactcatata tactttagat tgatttaaaa
     9541 cttcattttt aatttaaaag gatctaggtg aagatccttt ttgataatct catgaccaaa
     9601 atcccttaac gtgagttttc gttccactga gcgtcagacc ccgtagaaaa gatcaaagga
     9661 tcttcttgag atcctttttt tctgcgcgta atctgctgct tgcaaacaaa aaaaccaccg
     9721 ctaccagcgg tggtttgttt gccggatcaa gagctaccaa ctctttttcc gaaggtaact
     9781 ggcttcagca gagcgcagat accaaatact gttcttctag tgtagccgta gttaggccac
     9841 cacttcaaga actctgtagc accgcctaca tacctcgctc tgctaatcct gttaccagtg
     9901 gctgctgcca gtggcgataa gtcgtgtctt accgggttgg actcaagacg atagttaccg
     9961 gataaggcgc agcggtcggg ctgaacgggg ggttcgtgca cacagcccag cttggagcga
    10021 acgacctaca ccgaactgag atacctacag cgtgagctat gagaaagcgc cacgcttccc
    10081 gaagggagaa aggcggacag gtatccggta agcggcaggg tcggaacagg agagcgcacg
    10141 agggagcttc cagggggaaa cgcctggtat ctttatagtc ctgtcgggtt tcgccacctc
    10201 tgacttgagc gtcgattttt gtgatgctcg tcaggggggc ggagcctatg gaaaaacgcc
    10261 agcaacgcgg cctttttacg gttcctggcc tt
//
