LOCUS       sequence-377686-       10353 bp    DNA     circular SYN 15-DEC-2025
DEFINITION  Expresses spCas9 with a P2A junction to the puromycin resistance 
            gene directly fused to the thymidine kinase.
ACCESSION   .
VERSION     .
KEYWORDS    .
SOURCE      synthetic DNA construct
  ORGANISM  synthetic DNA construct
REFERENCE   1  (bases 1 to 10353)
  AUTHORS   Thomsen EA, Skipper KA, Andersen S, Haslund D, Skov TW, Mikkelsen JG
  TITLE     CRISPR-Cas9-directed gene tagging using a single integrase-defective
            lentiviral vector carrying a transposase-based Cas9 off switch.
  JOURNAL   Mol Ther Nucleic Acids. 2022 Aug 4;29:563-576. doi: 
            10.1016/j.omtn.2022.08.005. eCollection 2022 Sep 13.
  PUBMED    36090759
REFERENCE   2  (bases 1 to 10353)
  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: "Mol Ther 
            Nucleic Acids."; date: "2022-08-4"; volume: "29:563-576. doi"; 
            pages: " 10.1016/j.omtn.2022.08.005. eCollection 2022 Sep 13"
COMMENT     Sequence Label: pPBT/EFS-Cas9-PuroTk
FEATURES             Location/Qualifiers
     source          1..10353
                     /mol_type="other DNA"
                     /organism="synthetic DNA construct"
     promoter        24..128
                     /gene="bla"
                     /label=AmpR promoter
     CDS             join(129..197,198..989)
                     /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(347..366)
                     /label=Amp-R
                     /note="Ampicillin resistance gene, reverse primer"
     rep_origin      1160..1748
                     /direction=RIGHT
                     /label=ori
                     /note="high-copy-number ColE1/pMB1/pBR322/pUC origin of 
                     replication"
     primer_bind     1649..1668
                     /label=pBR322ori-F
                     /note="pBR322 origin, forward primer"
     primer_bind     1902..1919
                     /label=L4440
                     /note="L4440 vector, forward primer"
     protein_bind    2036..2057
                     /label=CAP binding site
                     /bound_moiety="E. coli catabolite activator protein"
                     /note="CAP binding activates transcription in the presence 
                     of cAMP."
     promoter        join(2072..2077,2078..2095,2096..2102)
                     /label=lac promoter
                     /note="promoter for the E. coli lac operon"
     protein_bind    2110..2126
                     /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)."
     primer_bind     2115..2137
                     /label=M13/pUC Reverse
                     /note="In lacZ gene"
     primer_bind     2134..2150
                     /label=M13 rev
                     /note="common sequencing primer, one of multiple similar 
                     variants"
     primer_bind     2134..2150
                     /label=M13 Reverse
                     /note="In lacZ gene. Also called M13-rev"
     repeat_region   2641..2675
                     /label=piggyBac left (5') inverted repeat
                     /note="piggyBac transposon-specific inverted terminal 
                     repeat sequence (ITR)"
     promoter        2999..3210
                     /label=EF-1-alpha core promoter
                     /note="core promoter for human elongation factor 
                     EF-1-alpha"
     regulatory      3229..3238
                     /label=Kozak sequence
                     /note="vertebrate consensus sequence for strong initiation 
                     of translation (Kozak, 1987)"
                     /regulatory_class="other"
     CDS             3235..7338
                     /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             3235..7338
                     /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             7339..7386
                     /codon_start=1
                     /product="bipartite nuclear localization signal from 
                     nucleoplasmin"
                     /label=nucleoplasmin NLS
                     /translation="KRPAATKKAGQAKKKK"
     CDS             7387..7410
                     /codon_start=1
                     /product="FLAG(R) epitope tag, followed by an enterokinase 
                     cleavage site"
                     /label=FLAG
                     /translation="DYKDDDDK"
     CDS             7420..7476
                     /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             7477..8070
                     /codon_start=1
                     /gene="pac from Streptomyces alboniger"
                     /product="puromycin N-acetyltransferase"
                     /label=PuroR
                     /note="confers resistance to puromycin"
                     /translation="TEYKPTVRLATRDDVPRAVRTLAAAFADYPATRHTVDPDRHIERV
                     TELQELFLTRVGLDIGKVWVADDGAAVAVWTTPESVEAGAVFAEIGPRMAELSGSRLAA
                     QQQMEGLLAPHRPKEPAWFLATVGVSPDHQGKGLGSAVVLPGVEAAERAGVPAFLETSA
                     PRNLPFYERLGFTVTADVEVPEGPRTWCMTRKPGA"
     primer_bind     7970..7990
                     /label=Puro-F
                     /note="Puromycin resistance gene, forward primer"
     CDS             8077..9072
                     /codon_start=1
                     /product="truncated herpes simplex virus thymidine kinase 
                     (Salomon et al., 1995)"
                     /label=Delta-TK
                     /note="confers sensitivity to nucleoside analogs such as 
                     acyclovir or ganciclovir"
                     /translation="MPTLLRVYIDGPHGMGKTTTTQLLVALGSRDDIVYVPEPMTYWQV
                     LGASETIANIYTTQHRLDQGEISAGDAAVVMTSAQITMGMPYAVTDAVLAPHIGGEAGS
                     SHAPPPALTLIFDRHPIAALLCYPAARYLMGSMTPQAVLAFVALIPPTLPGTNIVLGAL
                     PEDRHIDRLAKRQRPGERLDLAMLAAIRRVYGLLANTVRYLQGGGSWREDWGQLSGTAV
                     PPQGAEPQSNAGPRPHIGDTLFTLFRAPELLAPNGDLYNVFAWALDVLAKRLRPMHVFI
                     LDYDQSPAGCRDALLQLTSGMVQTHVTTPGSIPTICDLARTFAREMGEAN"
     primer_bind     complement(9099..9116)
                     /label=BGH-rev
                     /note="Bovine growth hormone terminator, reverse primer. 
                     Also called BGH reverse"
     polyA_signal    9105..9329
                     /label=bGH poly(A) signal
                     /note="bovine growth hormone polyadenylation signal"
     repeat_region   9546..9608
                     /label=piggyBac right (3') inverted repeat
                     /note="piggyBac transposon-specific inverted terminal 
                     repeat sequence (ITR)"
     primer_bind     complement(9738..9755)
                     /label=M13 Forward
                     /note="In lacZ gene. Also called M13-F20 or M13 (-21) 
                     Forward"
     primer_bind     complement(9738..9754)
                     /label=M13 fwd
                     /note="common sequencing primer, one of multiple similar 
                     variants"
     primer_bind     complement(9747..9769)
                     /label=M13/pUC Forward
                     /note="In lacZ gene"
     rep_origin      9896..10351
                     /direction=RIGHT
                     /label=M13 ori
                     /note="M13 bacteriophage origin of replication; arrow 
                     indicates direction of (+) strand synthesis"
     primer_bind     complement(9983..10002)
                     /label=F1ori-R
                     /note="F1 origin, reverse primer"
     primer_bind     10193..10214
                     /label=F1ori-F
                     /note="F1 origin, forward primer"
ORIGIN
        1 gtggcacttt tcggggaaat gtgcgcggaa cccctatttg tttatttttc taaatacatt
       61 caaatatgta tccgctcatg agacaataac cctgataaat gcttcaataa tattgaaaaa
      121 ggaagagtat gagtattcaa catttccgtg tcgcccttat tccctttttt gcggcatttt
      181 gccttcctgt ttttgctcac ccagaaacgc tggtgaaagt aaaagatgct gaagatcagt
      241 tgggtgcacg agtgggttac atcgaactgg atctcaacag cggtaagatc cttgagagtt
      301 ttcgccccga agaacgtttt ccaatgatga gcacttttaa agttctgcta tgtggcgcgg
      361 tattatcccg tattgacgcc gggcaagagc aactcggtcg ccgcatacac tattctcaga
      421 atgacttggt tgagtactca ccagtcacag aaaagcatct tacggatggc atgacagtaa
      481 gagaattatg cagtgctgcc ataaccatga gtgataacac tgcggccaac ttacttctga
      541 caacgatcgg aggaccgaag gagctaaccg cttttttgca caacatgggg gatcatgtaa
      601 ctcgccttga tcgttgggaa ccggagctga atgaagccat accaaacgac gagcgtgaca
      661 ccacgatgcc tgtagcaatg gcaacaacgt tgcgcaaact attaactggc gaactactta
      721 ctctagcttc ccggcaacaa ttaatagact ggatggaggc ggataaagtt gcaggaccac
      781 ttctgcgctc ggcccttccg gctggctggt ttattgctga taaatctgga gccggtgagc
      841 gtgggtctcg cggtatcatt gcagcactgg ggccagatgg taagccctcc cgtatcgtag
      901 ttatctacac gacggggagt caggcaacta tggatgaacg aaatagacag atcgctgaga
      961 taggtgcctc actgattaag cattggtaac tgtcagacca agtttactca tatatacttt
     1021 agattgattt aaaacttcat ttttaattta aaaggatcta ggtgaagatc ctttttgata
     1081 atctcatgac caaaatccct taacgtgagt tttcgttcca ctgagcgtca gaccccgtag
     1141 aaaagatcaa aggatcttct tgagatcctt tttttctgcg cgtaatctgc tgcttgcaaa
     1201 caaaaaaacc accgctacca gcggtggttt gtttgccgga tcaagagcta ccaactcttt
     1261 ttccgaaggt aactggcttc agcagagcgc agataccaaa tactgttctt ctagtgtagc
     1321 cgtagttagg ccaccacttc aagaactctg tagcaccgcc tacatacctc gctctgctaa
     1381 tcctgttacc agtggctgct gccagtggcg ataagtcgtg tcttaccggg ttggactcaa
     1441 gacgatagtt accggataag gcgcagcggt cgggctgaac ggggggttcg tgcacacagc
     1501 ccagcttgga gcgaacgacc tacaccgaac tgagatacct acagcgtgag ctatgagaaa
     1561 gcgccacgct tcccgaaggg agaaaggcgg acaggtatcc ggtaagcggc agggtcggaa
     1621 caggagagcg cacgagggag cttccagggg gaaacgcctg gtatctttat agtcctgtcg
     1681 ggtttcgcca cctctgactt gagcgtcgat ttttgtgatg ctcgtcaggg gggcggagcc
     1741 tatggaaaaa cgccagcaac gcggcctttt tacggttcct ggccttttgc tggccttttg
     1801 ctcacatgtt ctttcctgcg ttatcccctg attctgtgga taaccgtatt accgcctttg
     1861 agtgagctga taccgctcgc cgcagccgaa cgaccgagcg cagcgagtca gtgagcgagg
     1921 aagcggaaga gcgcccaata cgcaaaccgc ctctccccgc gcgttggccg attcattaat
     1981 gcagctggca cgacaggttt cccgactgga aagcgggcag tgagcgcaac gcaattaatg
     2041 tgagttagct cactcattag gcaccccagg ctttacactt tatgcttccg gctcgtatgt
     2101 tgtgtggaat tgtgagcgga taacaatttc acacaggaaa cagctatgac catgattacg
     2161 ccaagcgcgc ccgccgggta actcacgggg tatccatgtc catttctgcg gcatccagcc
     2221 aggatacccg tcctcgctga cgtaatatcc cagcgccgca ccgctgtcat taatctgcac
     2281 accggcacgg cagttccggc tgtcgccggt attgttcggg ttgctgatgc gcttcgggct
     2341 gaccatccgg aactgtgtcc ggaaaagccg cgacgaactg gtatcccagg tggcctgaac
     2401 gaacagttca ccgttaaagg cgtgcatggc cacaccttcc cgaatcatca tggtaaacgt
     2461 gcgttttcgc tcaacgtcaa tgcagcagca gtcatcctcg gcaaactctt tccatgccgc
     2521 ttcaacctcg cgggaaaagg cacgggcttc ttcctccccg atgcccagat agcgccagct
     2581 tgggcgatga ctgagccgga aaaaagaccc gacgatatga tcctgatgca gctagattaa
     2641 ccctagaaag atagtctgcg taaaattgac gcatgcattc ttgaaatatt gctctctctt
     2701 tctaaatagc gcgaatccgt cgctgtgcat ttaggacatc tcagtcgccg cttggagctc
     2761 ccgtgaggcg tgcttgtcaa tgcggtaagt gtcactgatt ttgaactata acgaccgcgt
     2821 gagtcaaaat gacgcatgat tatcttttac gtgactttta agatttaact catacgataa
     2881 ttatattgtt atttcatgtt ctacttacgt gataacttat tatatatata ttttcttgtt
     2941 atagatatca agctttaggt cttgaaagga gtgggaattg gctccggtgc ccgtcagtgg
     3001 gcagagcgca catcgcccac agtccccgag aagttggggg gaggggtcgg caattgatcc
     3061 ggtgcctaga gaaggtggcg cggggtaaac tgggaaagtg atgtcgtgta ctggctccgc
     3121 ctttttcccg agggtggggg agaaccgtat ataagtgcag tagtcgccgt gaacgttctt
     3181 tttcgcaacg ggtttgccgc cagaacacag gaccggttct agagcgctgc caccatggac
     3241 aagaagtaca gcatcggcct ggacatcggc accaactctg tgggctgggc cgtgatcacc
     3301 gacgagtaca aggtgcccag caagaaattc aaggtgctgg gcaacaccga ccggcacagc
     3361 atcaagaaga acctgatcgg agccctgctg ttcgacagcg gcgaaacagc cgaggccacc
     3421 cggctgaaga gaaccgccag aagaagatac accagacgga agaaccggat ctgctatctg
     3481 caagagatct tcagcaacga gatggccaag gtggacgaca gcttcttcca cagactggaa
     3541 gagtccttcc tggtggaaga ggataagaag cacgagcggc accccatctt cggcaacatc
     3601 gtggacgagg tggcctacca cgagaagtac cccaccatct accacctgag aaagaaactg
     3661 gtggacagca ccgacaaggc cgacctgcgg ctgatctatc tggccctggc ccacatgatc
     3721 aagttccggg gccacttcct gatcgagggc gacctgaacc ccgacaacag cgacgtggac
     3781 aagctgttca tccagctggt gcagacctac aaccagctgt tcgaggaaaa ccccatcaac
     3841 gccagcggcg tggacgccaa ggccatcctg tctgccagac tgagcaagag cagacggctg
     3901 gaaaatctga tcgcccagct gcccggcgag aagaagaatg gcctgttcgg aaacctgatt
     3961 gccctgagcc tgggcctgac ccccaacttc aagagcaact tcgacctggc cgaggatgcc
     4021 aaactgcagc tgagcaagga cacctacgac gacgacctgg acaacctgct ggcccagatc
     4081 ggcgaccagt acgccgacct gtttctggcc gccaagaacc tgtccgacgc catcctgctg
     4141 agcgacatcc tgagagtgaa caccgagatc accaaggccc ccctgagcgc ctctatgatc
     4201 aagagatacg acgagcacca ccaggacctg accctgctga aagctctcgt gcggcagcag
     4261 ctgcctgaga agtacaaaga gattttcttc gaccagagca agaacggcta cgccggctac
     4321 attgacggcg gagccagcca ggaagagttc tacaagttca tcaagcccat cctggaaaag
     4381 atggacggca ccgaggaact gctcgtgaag ctgaacagag aggacctgct gcggaagcag
     4441 cggaccttcg acaacggcag catcccccac cagatccacc tgggagagct gcacgccatt
     4501 ctgcggcggc aggaagattt ttacccattc ctgaaggaca accgggaaaa gatcgagaag
     4561 atcctgacct tccgcatccc ctactacgtg ggccctctgg ccaggggaaa cagcagattc
     4621 gcctggatga ccagaaagag cgaggaaacc atcaccccct ggaacttcga ggaagtggtg
     4681 gacaagggcg cttccgccca gagcttcatc gagcggatga ccaacttcga taagaacctg
     4741 cccaacgaga aggtgctgcc caagcacagc ctgctgtacg agtacttcac cgtgtataac
     4801 gagctgacca aagtgaaata cgtgaccgag ggaatgagaa agcccgcctt cctgagcggc
     4861 gagcagaaaa aggccatcgt ggacctgctg ttcaagacca accggaaagt gaccgtgaag
     4921 cagctgaaag aggactactt caagaaaatc gagtgcttcg actccgtgga aatctccggc
     4981 gtggaagatc ggttcaacgc ctccctgggc acataccacg atctgctgaa aattatcaag
     5041 gacaaggact tcctggacaa tgaggaaaac gaggacattc tggaagatat cgtgctgacc
     5101 ctgacactgt ttgaggacag agagatgatc gaggaacggc tgaaaaccta tgcccacctg
     5161 ttcgacgaca aagtgatgaa gcagctgaag cggcggagat acaccggctg gggcaggctg
     5221 agccggaagc tgatcaacgg catccgggac aagcagtccg gcaagacaat cctggatttc
     5281 ctgaagtccg acggcttcgc caacagaaac ttcatgcagc tgatccacga cgacagcctg
     5341 acctttaaag aggacatcca gaaagcccag gtgtccggcc agggcgatag cctgcacgag
     5401 cacattgcca atctggccgg cagccccgcc attaagaagg gcatcctgca gacagtgaag
     5461 gtggtggacg agctcgtgaa agtgatgggc cggcacaagc ccgagaacat cgtgatcgaa
     5521 atggccagag agaaccagac cacccagaag ggacagaaga acagccgcga gagaatgaag
     5581 cggatcgaag agggcatcaa agagctgggc agccagatcc tgaaagaaca ccccgtggaa
     5641 aacacccagc tgcagaacga gaagctgtac ctgtactacc tgcagaatgg gcgggatatg
     5701 tacgtggacc aggaactgga catcaaccgg ctgtccgact acgatgtgga ccatatcgtg
     5761 cctcagagct ttctgaagga cgactccatc gacaacaagg tgctgaccag aagcgacaag
     5821 aaccggggca agagcgacaa cgtgccctcc gaagaggtcg tgaagaagat gaagaactac
     5881 tggcggcagc tgctgaacgc caagctgatt acccagagaa agttcgacaa tctgaccaag
     5941 gccgagagag gcggcctgag cgaactggat aaggccggct tcatcaagag acagctggtg
     6001 gaaacccggc agatcacaaa gcacgtggca cagatcctgg actcccggat gaacactaag
     6061 tacgacgaga atgacaagct gatccgggaa gtgaaagtga tcaccctgaa gtccaagctg
     6121 gtgtccgatt tccggaagga tttccagttt tacaaagtgc gcgagatcaa caactaccac
     6181 cacgcccacg acgcctacct gaacgccgtc gtgggaaccg ccctgatcaa aaagtaccct
     6241 aagctggaaa gcgagttcgt gtacggcgac tacaaggtgt acgacgtgcg gaagatgatc
     6301 gccaagagcg agcaggaaat cggcaaggct accgccaagt acttcttcta cagcaacatc
     6361 atgaactttt tcaagaccga gattaccctg gccaacggcg agatccggaa gcggcctctg
     6421 atcgagacaa acggcgaaac cggggagatc gtgtgggata agggccggga ttttgccacc
     6481 gtgcggaaag tgctgagcat gccccaagtg aatatcgtga aaaagaccga ggtgcagaca
     6541 ggcggcttca gcaaagagtc tatcctgccc aagaggaaca gcgataagct gatcgccaga
     6601 aagaaggact gggaccctaa gaagtacggc ggcttcgaca gccccaccgt ggcctattct
     6661 gtgctggtgg tggccaaagt ggaaaagggc aagtccaaga aactgaagag tgtgaaagag
     6721 ctgctgggga tcaccatcat ggaaagaagc agcttcgaga agaatcccat cgactttctg
     6781 gaagccaagg gctacaaaga agtgaaaaag gacctgatca tcaagctgcc taagtactcc
     6841 ctgttcgagc tggaaaacgg ccggaagaga atgctggcct ctgccggcga actgcagaag
     6901 ggaaacgaac tggccctgcc ctccaaatat gtgaacttcc tgtacctggc cagccactat
     6961 gagaagctga agggctcccc cgaggataat gagcagaaac agctgtttgt ggaacagcac
     7021 aagcactacc tggacgagat catcgagcag atcagcgagt tctccaagag agtgatcctg
     7081 gccgacgcta atctggacaa agtgctgtcc gcctacaaca agcaccggga taagcccatc
     7141 agagagcagg ccgagaatat catccacctg tttaccctga ccaatctggg agcccctgcc
     7201 gccttcaagt actttgacac caccatcgac cggaagaggt acaccagcac caaagaggtg
     7261 ctggacgcca ccctgatcca ccagagcatc accggcctgt acgagacacg gatcgacctg
     7321 tctcagctgg gaggcgacaa gcgacctgcc gccacaaaga aggctggaca ggctaagaag
     7381 aagaaagatt acaaagacga tgacgataag ggatccggcg caacaaactt ctctctgctg
     7441 aaacaagccg gagatgtcga agagaatcct ggaccgaccg agtacaagcc cacggtgcgc
     7501 ctcgccaccc gcgacgacgt ccccagggcc gtacgcaccc tcgccgccgc gttcgccgac
     7561 taccccgcca cgcgccacac cgtcgatccg gaccgccaca tcgagcgggt caccgagctg
     7621 caagaactct tcctcacgcg cgtcgggctc gacatcggca aggtgtgggt cgcggacgac
     7681 ggcgccgcgg tggcggtctg gaccacgccg gagagcgtcg aagcgggggc ggtgttcgcc
     7741 gagatcggcc cgcgcatggc cgagttgagc ggttcccggc tggccgcgca gcaacagatg
     7801 gaaggcctcc tggcgccgca ccggcccaag gagcccgcgt ggttcctggc caccgtcggc
     7861 gtctcgcccg accaccaggg caagggtctg ggcagcgccg tcgtgctccc cggagtggag
     7921 gcggccgagc gcgccggggt gcccgccttc ctggagacct ccgcgccccg caacctcccc
     7981 ttctacgagc ggctcggctt caccgtcacc gccgacgtcg aggtgcccga aggaccgcgc
     8041 acctggtgca tgacccgcaa gcccggtgcc ggatccatgc ccacgctact gcgggtttat
     8101 atagacggtc ctcacgggat ggggaaaacc accaccacgc aactgctggt ggccctgggt
     8161 tcgcgcgacg atatcgtcta cgtacccgag ccgatgactt actggcaggt gctgggggct
     8221 tccgagacaa tcgcgaacat ctacaccaca caacaccgcc tcgaccaggg tgagatatcg
     8281 gccggggacg cggcggtggt aatgacaagc gcccagataa caatgggcat gccttatgcc
     8341 gtgaccgacg ccgttctggc tcctcatatc gggggggagg ctgggagctc acatgccccg
     8401 cccccggccc tcaccctcat cttcgaccgc catcccatcg ccgccctcct gtgctacccg
     8461 gccgcgcgat accttatggg cagcatgacc ccccaggccg tgctggcgtt cgtggccctc
     8521 atcccgccga ccttgcccgg cacaaacatc gtgttggggg cccttccgga ggacagacac
     8581 atcgaccgcc tggccaaacg ccagcgcccc ggcgagcggc ttgacctggc tatgctggcc
     8641 gcgattcgcc gcgtttacgg gctgcttgcc aatacggtgc ggtatctgca gggcggcggg
     8701 tcgtggcggg aggattgggg acagctttcg gggacggccg tgccgcccca gggtgccgag
     8761 ccccagagca acgcgggccc acgaccccat atcggggaca cgttatttac cctgtttcgg
     8821 gcccccgagt tgctggcccc caacggcgac ctgtacaacg tgtttgcctg ggccttggac
     8881 gtcttggcca aacgcctccg tcccatgcac gtctttatcc tggattacga ccaatcgccc
     8941 gccggctgcc gggacgccct gctgcaactt acctccggga tggtccagac ccacgtcacc
     9001 acccccggct ccataccgac gatctgcgac ctggcgcgca cgtttgcccg ggagatgggg
     9061 gaggctaact gacatatgta gatcccccgg gggatcagcc tcgactgtgc cttctagttg
     9121 ccagccatct gttgtttgcc cctcccccgt gccttccttg accctggaag gtgccactcc
     9181 cactgtcctt tcctaataaa atgaggaaat tgcatcgcat tgtctgagta ggtgtcattc
     9241 tattctgggg ggtggggtgg ggcaggacag caagggggag gattgggaag acaatagcag
     9301 gcatgctggg gatgcggtgg gctctatggg cggccgctct agaactagtg ccccgggctg
     9361 caggaattcg ataaaagttt tgttacttta tagaagaaat tttgagtttt tgtttttttt
     9421 aataaataaa taaacataaa taaattgttt gttgaattta ttattagtat gtaagtgtaa
     9481 atataataaa acttaatatc tattcaaatt aataaataaa cctcgatata cagaccgata
     9541 aaacacatgc gtcaatttta cacatgatta tctttaacgt acgtcacaat atgattatct
     9601 ttctagggtt aatctagctg cgtgttctgc agcgtgtcga gcatcttcat ctgctccatc
     9661 acgctgtaaa acacatttgc accgcgagtc tgcccgtcct ccacgggttc aaaaacgtga
     9721 atgaacgagg cgcgctcact ggccgtcgtt ttacaacgtc gtgactggga aaaccctggc
     9781 gttacccaac ttaatcgcct tgcagcacat ccccctttcg ccagctggcg taatagcgaa
     9841 gaggcccgca ccgatcgccc ttcccaacag ttgcgcagcc tgaatggcga atgggacgcg
     9901 ccctgtagcg gcgcattaag cgcggcgggt gtggtggtta cgcgcagcgt gaccgctaca
     9961 cttgccagcg ccctagcgcc cgctcctttc gctttcttcc cttcctttct cgccacgttc
    10021 gccggctttc cccgtcaagc tctaaatcgg gggctccctt tagggttccg atttagtgct
    10081 ttacggcacc tcgaccccaa aaaacttgat tagggtgatg gttcacgtag tgggccatcg
    10141 ccctgataga cggtttttcg ccctttgacg ttggagtcca cgttctttaa tagtggactc
    10201 ttgttccaaa ctggaacaac actcaaccct atctcggtct attcttttga tttataaggg
    10261 attttgccga tttcggccta ttggttaaaa aatgagctga tttaacaaaa atttaacgcg
    10321 aattttaaca aaatattaac gcttacaatt tag
//
