LOCUS       sequence-423617-       10961 bp    DNA     circular SYN 15-DEC-2025
DEFINITION  Gene expression by Piggybac transposase in mammalian cells.
ACCESSION   .
VERSION     .
KEYWORDS    .
SOURCE      synthetic DNA construct
  ORGANISM  synthetic DNA construct
REFERENCE   1  (bases 1 to 10961)
  TITLE     Yoshimatsu Lab plasmids 2023
REFERENCE   2  (bases 1 to 10961)
  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"
COMMENT     Sequence Label: pPB-spCas9-pNeo
FEATURES             Location/Qualifiers
     source          1..10961
                     /mol_type="other DNA"
                     /organism="synthetic DNA construct"
     rep_origin      complement(30..458)
                     /direction=LEFT
                     /label=f1 ori
                     /note="f1 bacteriophage origin of replication; arrow 
                     indicates direction of (+) strand synthesis"
     primer_bind     complement(140..161)
                     /label=F1ori-F
                     /note="F1 origin, forward primer"
     primer_bind     352..371
                     /label=F1ori-R
                     /note="F1 origin, reverse primer"
     primer_bind     585..607
                     /label=M13/pUC Forward
                     /note="In lacZ gene"
     primer_bind     599..616
                     /label=M13 Forward
                     /note="In lacZ gene. Also called M13-F20 or M13 (-21) 
                     Forward"
     primer_bind     600..616
                     /label=M13 fwd
                     /note="common sequencing primer, one of multiple similar 
                     variants"
     primer_bind     623..642
                     /label=T7
                     /note="T7 promoter, forward primer"
     promoter        623..641
                     /label=T7 promoter
                     /note="promoter for bacteriophage T7 RNA polymerase"
     repeat_region   660..694
                     /label=piggyBac left (5') inverted repeat
                     /note="piggyBac transposon-specific inverted terminal 
                     repeat sequence (ITR)"
     promoter        1014..1262
                     /label=U6 promoter
                     /note="RNA polymerase III promoter for human U6 snRNA 
                     (Domitrovich & Kunkel, 2003)"
     primer_bind     1014..1034
                     /label=hU6-F
                     /note="Human U6 promoter, forward primer"
     primer_bind     1185..1204
                     /label=LKO.1 5'
                     /note="Human U6 promoter, forward primer"
     misc_RNA        1281..1356
                     /label=gRNA scaffold
                     /note="guide RNA scaffold for the Streptococcus pyogenes 
                     CRISPR/Cas9 system"
     enhancer        1453..1738
                     /label=CMV enhancer
                     /note="human cytomegalovirus immediate early enhancer; 
                     contains an 18-bp deletion relative to the standard CMV 
                     enhancer"
     promoter        1740..2017
                     /label=chicken beta-actin promoter
     intron          2018..2245
                     /label=hybrid intron
                     /note="hybrid between chicken beta-actin (CBA) and minute 
                     virus of mice (MMV) introns (Gray et al., 2011)"
     regulatory      2257..2266
                     /label=Kozak sequence
                     /note="vertebrate consensus sequence for strong initiation 
                     of translation (Kozak, 1987)"
                     /regulatory_class="other"
     CDS             2266..2331
                     /codon_start=1
                     /product="three tandem FLAG(R) epitope tags, followed by an
                     enterokinase cleavage site"
                     /label=3xFLAG
                     /translation="DYKDHDGDYKDHDIDYKDDDDK"
     CDS             2338..2358
                     /codon_start=1
                     /product="nuclear localization signal of SV40 (simian virus
                     40) large T antigen"
                     /label=SV40 NLS
                     /translation="PKKKRKV"
     CDS             2383..6483
                     /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             2383..6483
                     /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             6484..6531
                     /codon_start=1
                     /product="bipartite nuclear localization signal from 
                     nucleoplasmin"
                     /label=nucleoplasmin NLS
                     /translation="KRPAATKKAGQAKKKK"
     primer_bind     complement(6559..6576)
                     /label=BGH-rev
                     /note="Bovine growth hormone terminator, reverse primer. 
                     Also called BGH reverse"
     polyA_signal    6565..6772
                     /label=bGH poly(A) signal
                     /note="bovine growth hormone polyadenylation signal"
     promoter        6957..7456
                     /label=PGK promoter
                     /note="mouse phosphoglycerate kinase 1 promoter"
     primer_bind     complement(6992..7015)
                     /label=MSCV-rev
                     /note="Murine stem cell virus, reverse primer"
     primer_bind     7389..7408
                     /label=mPGK-F
                     /note="Mouse PGK promoter, forward primer"
     CDS             7470..8273
                     /codon_start=1
                     /gene="aph(3')-II (or nptII)"
                     /product="aminoglycoside phosphotransferase from Tn5"
                     /label=NeoR/KanR
                     /note="confers resistance to neomycin, kanamycin, and G418 
                     (Geneticin(R))"
                     /translation="MGSAIEQDGLHAGSPAAWVERLFGYDWAQQTIGCSDAAVFRLSAQ
                     GRPVLFVKTDLSGALNELQDEAARLSWLATTGVPCAAVLDVVTEAGRDWLLLGEVPGQD
                     LLSSHLAPAEKVSIMADAMRRLHTLDPATCPFDHQAKHRIERARTRMEAGLVDQDDLDE
                     EHQGLAPAELFARLKARMPDGDDLVVTHGDACLPNIMVENGRFSGFIDCGRLGVADRYQ
                     DIALATRDIAEELGGEWADRFLVLYGIAAPDSQRIAFYRLLDEFF"
     primer_bind     complement(7533..7552)
                     /label=Neo-R
                     /note="Neomycin resistance gene, reverse primer"
     primer_bind     8143..8162
                     /label=Neo-F
                     /note="Neomycin resistance gene, forward primer"
     repeat_region   8687..8749
                     /label=piggyBac right (3') inverted repeat
                     /note="piggyBac transposon-specific inverted terminal 
                     repeat sequence (ITR)"
     promoter        complement(8773..8791)
                     /label=T3 promoter
                     /note="promoter for bacteriophage T3 RNA polymerase"
     primer_bind     complement(8812..8828)
                     /label=M13 rev
                     /note="common sequencing primer, one of multiple similar 
                     variants"
     primer_bind     complement(8812..8828)
                     /label=M13 Reverse
                     /note="In lacZ gene. Also called M13-rev"
     primer_bind     complement(8825..8847)
                     /label=M13/pUC Reverse
                     /note="In lacZ gene"
     protein_bind    8836..8852
                     /label=lac operator
                     /bound_moiety="lac repressor encoded by lacI"
                     /note="The lac repressor binds to the lac operator to 
                     inhibit transcription in E. coli. This inhibition can be 
                     relieved by adding lactose or 
                     isopropyl-beta-D-thiogalactopyranoside (IPTG)."
     promoter        complement(join(8860..8866,8867..8884,8885..8890))
                     /label=lac promoter
                     /note="promoter for the E. coli lac operon"
     protein_bind    8905..8926
                     /label=CAP binding site
                     /bound_moiety="E. coli catabolite activator protein"
                     /note="CAP binding activates transcription in the presence 
                     of cAMP."
     primer_bind     complement(9043..9060)
                     /label=L4440
                     /note="L4440 vector, forward primer"
     rep_origin      complement(9214..9802)
                     /direction=LEFT
                     /label=ori
                     /note="high-copy-number ColE1/pMB1/pBR322/pUC origin of 
                     replication"
     primer_bind     complement(9294..9313)
                     /label=pBR322ori-F
                     /note="pBR322 origin, forward primer"
     CDS             complement(join(9973..10764,10765..10833))
                     /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     10596..10615
                     /label=Amp-R
                     /note="Ampicillin resistance gene, reverse primer"
     promoter        complement(10834..10938)
                     /gene="bla"
                     /label=AmpR promoter
ORIGIN
        1 ctaaattgta agcgttaata ttttgttaaa attcgcgtta aatttttgtt aaatcagctc
       61 attttttaac caataggccg aaatcggcaa aatcccttat aaatcaaaag aatagaccga
      121 gatagggttg agtgttgttc cagtttggaa caagagtcca ctattaaaga acgtggactc
      181 caacgtcaaa gggcgaaaaa ccgtctatca gggcgatggc ccactacgtg aaccatcacc
      241 ctaatcaagt tttttggggt cgaggtgccg taaagcacta aatcggaacc ctaaagggag
      301 cccccgattt agagcttgac ggggaaagcc ggcgaacgtg gcgagaaagg aagggaagaa
      361 agcgaaagga gcgggcgcta gggcgctggc aagtgtagcg gtcacgctgc gcgtaaccac
      421 cacacccgcc gcgcttaatg cgccgctaca gggcgcgtcc cattcgccat tcaggctgcg
      481 caactgttgg gaagggcgat cggtgcgggc ctcttcgcta ttacgccagc tggcgaaagg
      541 gggatgtgct gcaaggcgat taagttgggt aacgccaggg ttttcccagt cacgacgttg
      601 taaaacgacg gccagtgaat tgtaatacga ctcactatag ggcgaattgg gtaccttaac
      661 cctagaaaga tagtctgcgt aaaattgacg catgcattct tgaaatattg ctctctcttt
      721 ctaaatagcg cgaatccgtc gctgtgcatt taggacatct cagtcgccgc ttggagctcc
      781 cgtgaggcgt gcttgtcaat gaggtaagtg tcactgattt tgaactataa cgaccgcgtg
      841 agtcaaaatg acgcatgatt atcttttacg tgacttttaa gatttaactc atacgataat
      901 tatattgtta tttcatgttc tacttacgtg ataacttatt atatatatat tttcttgtta
      961 tagatgatcc actagtaacg gccgccagtg tgctggaatt cgcggcccac atggagggcc
     1021 tatttcccat gattccttca tatttgcata tacgatacaa ggctgttaga gagataattg
     1081 gaattaattt gactgtaaac acaaagatat tagtacaaaa tacgtgacgt agaaagtaat
     1141 aatttcttgg gtagtttgca gttttaaaat tatgttttaa aatggactat catatgctta
     1201 ccgtaacttg aaagtatttc gatttcttgg ctttatatat cttgtggaaa ggacgaaaca
     1261 ccgggtcttc gagaagacct gttttagagc tagaaatagc aagttaaaat aaggctagtc
     1321 cgttatcaac ttgaaaaagt ggcaccgagt cggtgctttt ttgttttaga gctagaaata
     1381 gcaagttaaa ataaggctag tccgttttta gcgcgtgcgc caattctgca gacaaatggc
     1441 tctagaggta cccgttacat aacttacggt aaatggcccg cctggctgac cgcccaacga
     1501 cccccgccca ttgacgtcaa tagtaacgcc aatagggact ttccattgac gtcaatgggt
     1561 ggagtattta cggtaaactg cccacttggc agtacatcaa gtgtatcata tgccaagtac
     1621 gccccctatt gacgtcaatg acggtaaatg gcccgcctgg cattgtgccc agtacatgac
     1681 cttatgggac tttcctactt ggcagtacat ctacgtatta gtcatcgcta ttaccatggt
     1741 cgaggtgagc cccacgttct gcttcactct ccccatctcc cccccctccc cacccccaat
     1801 tttgtattta tttatttttt aattattttg tgcagcgatg ggggcggggg gggggggggg
     1861 gcgcgcgcca ggcggggcgg ggcggggcga ggggcggggc ggggcgaggc ggagaggtgc
     1921 ggcggcagcc aatcagagcg gcgcgctccg aaagtttcct tttatggcga ggcggcggcg
     1981 gcggcggccc tataaaaagc gaagcgcgcg gcgggcggga gtcgctgcgc gctgccttcg
     2041 ccccgtgccc cgctccgccg ccgcctcgcg ccgcccgccc cggctctgac tgaccgcgtt
     2101 actcccacag gtgagcgggc gggacggccc ttctcctccg ggctgtaatt agctgagcaa
     2161 gaggtaaggg tttaagggat ggttggttgg tggggtatta atgtttaatt acctggagca
     2221 cctgcctgaa atcacttttt ttcaggttgg accggtgcca ccatggacta taaggaccac
     2281 gacggagact acaaggatca tgatattgat tacaaagacg atgacgataa gatggcccca
     2341 aagaagaagc ggaaggtcgg tatccacgga gtcccagcag ccgacaagaa gtacagcatc
     2401 ggcctggaca tcggcaccaa ctctgtgggc tgggccgtga tcaccgacga gtacaaggtg
     2461 cccagcaaga aattcaaggt gctgggcaac accgaccggc acagcatcaa gaagaacctg
     2521 atcggagccc tgctgttcga cagcggcgaa acagccgagg ccacccggct gaagagaacc
     2581 gccagaagaa gatacaccag acggaagaac cggatctgct atctgcaaga gatcttcagc
     2641 aacgagatgg ccaaggtgga cgacagcttc ttccacagac tggaagagtc cttcctggtg
     2701 gaagaggata agaagcacga gcggcacccc atcttcggca acatcgtgga cgaggtggcc
     2761 taccacgaga agtaccccac catctaccac ctgagaaaga aactggtgga cagcaccgac
     2821 aaggccgacc tgcggctgat ctatctggcc ctggcccaca tgatcaagtt ccggggccac
     2881 ttcctgatcg agggcgacct gaaccccgac aacagcgacg tggacaagct gttcatccag
     2941 ctggtgcaga cctacaacca gctgttcgag gaaaacccca tcaacgccag cggcgtggac
     3001 gccaaggcca tcctgtctgc cagactgagc aagagcagac ggctggaaaa tctgatcgcc
     3061 cagctgcccg gcgagaagaa gaatggcctg ttcggaaacc tgattgccct gagcctgggc
     3121 ctgaccccca acttcaagag caacttcgac ctggccgagg atgccaaact gcagctgagc
     3181 aaggacacct acgacgacga cctggacaac ctgctggccc agatcggcga ccagtacgcc
     3241 gacctgtttc tggccgccaa gaacctgtcc gacgccatcc tgctgagcga catcctgaga
     3301 gtgaacaccg agatcaccaa ggcccccctg agcgcctcta tgatcaagag atacgacgag
     3361 caccaccagg acctgaccct gctgaaagct ctcgtgcggc agcagctgcc tgagaagtac
     3421 aaagagattt tcttcgacca gagcaagaac ggctacgccg gctacattga cggcggagcc
     3481 agccaggaag agttctacaa gttcatcaag cccatcctgg aaaagatgga cggcaccgag
     3541 gaactgctcg tgaagctgaa cagagaggac ctgctgcgga agcagcggac cttcgacaac
     3601 ggcagcatcc cccaccagat ccacctggga gagctgcacg ccattctgcg gcggcaggaa
     3661 gatttttacc cattcctgaa ggacaaccgg gaaaagatcg agaagatcct gaccttccgc
     3721 atcccctact acgtgggccc tctggccagg ggaaacagca gattcgcctg gatgaccaga
     3781 aagagcgagg aaaccatcac cccctggaac ttcgaggaag tggtggacaa gggcgcttcc
     3841 gcccagagct tcatcgagcg gatgaccaac ttcgataaga acctgcccaa cgagaaggtg
     3901 ctgcccaagc acagcctgct gtacgagtac ttcaccgtgt ataacgagct gaccaaagtg
     3961 aaatacgtga ccgagggaat gagaaagccc gccttcctga gcggcgagca gaaaaaggcc
     4021 atcgtggacc tgctgttcaa gaccaaccgg aaagtgaccg tgaagcagct gaaagaggac
     4081 tacttcaaga aaatcgagtg cttcgactcc gtggaaatct ccggcgtgga agatcggttc
     4141 aacgcctccc tgggcacata ccacgatctg ctgaaaatta tcaaggacaa ggacttcctg
     4201 gacaatgagg aaaacgagga cattctggaa gatatcgtgc tgaccctgac actgtttgag
     4261 gacagagaga tgatcgagga acggctgaaa acctatgccc acctgttcga cgacaaagtg
     4321 atgaagcagc tgaagcggcg gagatacacc ggctggggca ggctgagccg gaagctgatc
     4381 aacggcatcc gggacaagca gtccggcaag acaatcctgg atttcctgaa gtccgacggc
     4441 ttcgccaaca gaaacttcat gcagctgatc cacgacgaca gcctgacctt taaagaggac
     4501 atccagaaag cccaggtgtc cggccagggc gatagcctgc acgagcacat tgccaatctg
     4561 gccggcagcc ccgccattaa gaagggcatc ctgcagacag tgaaggtggt ggacgagctc
     4621 gtgaaagtga tgggccggca caagcccgag aacatcgtga tcgaaatggc cagagagaac
     4681 cagaccaccc agaagggaca gaagaacagc cgcgagagaa tgaagcggat cgaagagggc
     4741 atcaaagagc tgggcagcca gatcctgaaa gaacaccccg tggaaaacac ccagctgcag
     4801 aacgagaagc tgtacctgta ctacctgcag aatgggcggg atatgtacgt ggaccaggaa
     4861 ctggacatca accggctgtc cgactacgat gtggaccata tcgtgcctca gagctttctg
     4921 aaggacgact ccatcgacaa caaggtgctg accagaagcg acaagaaccg gggcaagagc
     4981 gacaacgtgc cctccgaaga ggtcgtgaag aagatgaaga actactggcg gcagctgctg
     5041 aacgccaagc tgattaccca gagaaagttc gacaatctga ccaaggccga gagaggcggc
     5101 ctgagcgaac tggataaggc cggcttcatc aagagacagc tggtggaaac ccggcagatc
     5161 acaaagcacg tggcacagat cctggactcc cggatgaaca ctaagtacga cgagaatgac
     5221 aagctgatcc gggaagtgaa agtgatcacc ctgaagtcca agctggtgtc cgatttccgg
     5281 aaggatttcc agttttacaa agtgcgcgag atcaacaact accaccacgc ccacgacgcc
     5341 tacctgaacg ccgtcgtggg aaccgccctg atcaaaaagt accctaagct ggaaagcgag
     5401 ttcgtgtacg gcgactacaa ggtgtacgac gtgcggaaga tgatcgccaa gagcgagcag
     5461 gaaatcggca aggctaccgc caagtacttc ttctacagca acatcatgaa ctttttcaag
     5521 accgagatta ccctggccaa cggcgagatc cggaagcggc ctctgatcga gacaaacggc
     5581 gaaaccgggg agatcgtgtg ggataagggc cgggattttg ccaccgtgcg gaaagtgctg
     5641 agcatgcccc aagtgaatat cgtgaaaaag accgaggtgc agacaggcgg cttcagcaaa
     5701 gagtctatcc tgcccaagag gaacagcgat aagctgatcg ccagaaagaa ggactgggac
     5761 cctaagaagt acggcggctt cgacagcccc accgtggcct attctgtgct ggtggtggcc
     5821 aaagtggaaa agggcaagtc caagaaactg aagagtgtga aagagctgct ggggatcacc
     5881 atcatggaaa gaagcagctt cgagaagaat cccatcgact ttctggaagc caagggctac
     5941 aaagaagtga aaaaggacct gatcatcaag ctgcctaagt actccctgtt cgagctggaa
     6001 aacggccgga agagaatgct ggcctctgcc ggcgaactgc agaagggaaa cgaactggcc
     6061 ctgccctcca aatatgtgaa cttcctgtac ctggccagcc actatgagaa gctgaagggc
     6121 tcccccgagg ataatgagca gaaacagctg tttgtggaac agcacaagca ctacctggac
     6181 gagatcatcg agcagatcag cgagttctcc aagagagtga tcctggccga cgctaatctg
     6241 gacaaagtgc tgtccgccta caacaagcac cgggataagc ccatcagaga gcaggccgag
     6301 aatatcatcc acctgtttac cctgaccaat ctgggagccc ctgccgcctt caagtacttt
     6361 gacaccacca tcgaccggaa gaggtacacc agcaccaaag aggtgctgga cgccaccctg
     6421 atccaccaga gcatcaccgg cctgtacgag acacggatcg acctgtctca gctgggaggc
     6481 gacaaaaggc cggcggccac gaaaaaggcc ggccaggcaa aaaagaaaaa ggaattctaa
     6541 ctagagctcg ctgatcagcc tcgactgtgc cttctagttg ccagccatct gttgtttgcc
     6601 cctcccccgt gccttccttg accctggaag gtgccactcc cactgtcctt tcctaataaa
     6661 atgaggaaat tgcatcgcat tgtctgagta ggtgtcattc tattctgggg ggtggggtgg
     6721 ggcaggacag caagggggag gattgggaag agaatagcag gcatgctggg gagcggccgc
     6781 tttgtcgaca agctttaatg cggtagttta tcacagttaa attgctaacg cagtcaggca
     6841 ccgtgtatga aatctaacaa tgcgctcatc gtcatcctcg gcaccgtcac cctggatgct
     6901 gtaggcatag gcttggttat gccggtactg ccgggcctct tgcggggaat tctaccgggt
     6961 aggggaggcg cttttcccaa ggcagtctgg agcatgcgct ttagcagccc cgctgggcac
     7021 ttggcgctac acaagtggcc tctggcctcg cacacattcc acatccaccg gtaggcgcca
     7081 accggctccg ttctttggtg gccccttcgc gccaccttct actcctcccc tagtcaggaa
     7141 gttccccccc gccccgcagc tcgcgtcgtg caggacgtga caaatggaag tagcacgtct
     7201 cactagtctc gtgcagatgg acagcaccgc tgagcaatgg aagcgggtag gcctttgggg
     7261 cagcggccaa tagcagcttt gctccttcgc tttctgggct cagaggctgg gaaggggtgg
     7321 gtccgggggc gggctcaggg gcgggctcag gggcggggcg ggcgcccgaa ggtcctccgg
     7381 aggcccggca ttctgcacgc ttcaaaagcg cacgtctgcc gcgctgttct cctcttcctc
     7441 atctccgggc ctttcgacct gcagccaata tgggatcggc cattgaacaa gatggattgc
     7501 acgcaggttc tccggccgct tgggtggaga ggctattcgg ctatgactgg gcacaacaga
     7561 caatcggctg ctctgatgcc gccgtgttcc ggctgtcagc gcaggggcgc ccggttcttt
     7621 ttgtcaagac cgacctgtcc ggtgccctga atgaactgca ggacgaggca gcgcggctat
     7681 cgtggctggc cacgacgggc gttccttgcg cagctgtgct cgacgttgtc actgaagcgg
     7741 gaagggactg gctgctattg ggcgaagtgc cggggcagga tctcctgtca tctcaccttg
     7801 ctcctgccga gaaagtatcc atcatggctg atgcaatgcg gcggctgcat acgcttgatc
     7861 cggctacctg cccattcgac caccaagcga aacatcgcat cgagcgagca cgtactcgga
     7921 tggaagccgg tcttgtcgat caggatgatc tggacgaaga gcatcagggg ctcgcgccag
     7981 ccgaactgtt cgccaggctc aaggcgcgca tgcccgacgg cgatgatctc gtcgtgaccc
     8041 atggcgatgc ctgcttgccg aatatcatgg tggaaaatgg ccgcttttct ggattcatcg
     8101 actgtggccg gctgggtgtg gcggaccgct atcaggacat agcgttggct acccgtgata
     8161 ttgctgaaga gcttggcggc gaatgggctg accgcttcct cgtgctttac ggtatcgccg
     8221 ctcccgattc gcagcgcatc gccttctatc gccttcttga cgagttcttc tgaggggatc
     8281 gatccgctgt aagtctgcag aaattgatga tctattaaac aataaagatg tccactaaaa
     8341 tggaagtttt tcctgtcata ctttgttaag aagggtgaga acagagtacc tacattttga
     8401 atggaaggat tggagctacg ggggtggggg tggggtggga ttagataaat gcctgctctt
     8461 tactgaaggc tctttactat tgctttatga taatgtttca tagttggata tcagatcctt
     8521 tgttacttta tagaagaaat tttgagtttt tgtttttttt taataaataa ataaacataa
     8581 ataaattgtt tgttgaattt attattagta tgtaagtgta aatataataa aacttaatat
     8641 ctattcaaat taataaataa acctcgatat acagaccgat aaaacacatg cgtcaatttt
     8701 acgcatgatt atctttaacg tacgtcacaa tatgattatc tttctagggt taaagagctc
     8761 cagcttttgt tccctttagt gagggttaat ttcgagcttg gcgtaatcat ggtcatagct
     8821 gtttcctgtg tgaaattgtt atccgctcac aattccacac aacatacgag ccggaagcat
     8881 aaagtgtaaa gcctggggtg cctaatgagt gagctaactc acattaattg cgttgcgctc
     8941 actgcccgct ttccagtcgg gaaacctgtc gtgccagctg cattaatgaa tcggccaacg
     9001 cgcggggaga ggcggtttgc gtattgggcg ctcttccgct tcctcgctca ctgactcgct
     9061 gcgctcggtc gttcggctgc ggcgagcggt atcagctcac tcaaaggcgg taatacggtt
     9121 atccacagaa tcaggggata acgcaggaaa gaacatgtga gcaaaaggcc agcaaaaggc
     9181 caggaaccgt aaaaaggccg cgttgctggc gtttttccat aggctccgcc cccctgacga
     9241 gcatcacaaa aatcgacgct caagtcagag gtggcgaaac ccgacaggac tataaagata
     9301 ccaggcgttt ccccctggaa gctccctcgt gcgctctcct gttccgaccc tgccgcttac
     9361 cggatacctg tccgcctttc tcccttcggg aagcgtggcg ctttctcata gctcacgctg
     9421 taggtatctc agttcggtgt aggtcgttcg ctccaagctg ggctgtgtgc acgaaccccc
     9481 cgttcagccc gaccgctgcg ccttatccgg taactatcgt cttgagtcca acccggtaag
     9541 acacgactta tcgccactgg cagcagccac tggtaacagg attagcagag cgaggtatgt
     9601 aggcggtgct acagagttct tgaagtggtg gcctaactac ggctacacta gaagaacagt
     9661 atttggtatc tgcgctctgc tgaagccagt taccttcgga aaaagagttg gtagctcttg
     9721 atccggcaaa caaaccaccg ctggtagcgg tggttttttt gtttgcaagc agcagattac
     9781 gcgcagaaaa aaaggatctc aagaagatcc tttgatcttt tctacggggt ctgacgctca
     9841 gtggaacgaa aactcacgtt aagggatttt ggtcatgaga ttatcaaaaa ggatcttcac
     9901 ctagatcctt ttaaattaaa aatgaagttt taaatcaatc taaagtatat atgagtaaac
     9961 ttggtctgac agttaccaat gcttaatcag tgaggcacct atctcagcga tctgtctatt
    10021 tcgttcatcc atagttgcct gactccccgt cgtgtagata actacgatac gggagggctt
    10081 accatctggc cccagtgctg caatgatacc gcgagaccca cgctcaccgg ctccagattt
    10141 atcagcaata aaccagccag ccggaagggc cgagcgcaga agtggtcctg caactttatc
    10201 cgcctccatc cagtctatta attgttgccg ggaagctaga gtaagtagtt cgccagttaa
    10261 tagtttgcgc aacgttgttg ccattgctac aggcatcgtg gtgtcacgct cgtcgtttgg
    10321 tatggcttca ttcagctccg gttcccaacg atcaaggcga gttacatgat cccccatgtt
    10381 gtgcaaaaaa gcggttagct ccttcggtcc tccgatcgtt gtcagaagta agttggccgc
    10441 agtgttatca ctcatggtta tggcagcact gcataattct cttactgtca tgccatccgt
    10501 aagatgcttt tctgtgactg gtgagtactc aaccaagtca ttctgagaat agtgtatgcg
    10561 gcgaccgagt tgctcttgcc cggcgtcaat acgggataat accgcgccac atagcagaac
    10621 tttaaaagtg ctcatcattg gaaaacgttc ttcggggcga aaactctcaa ggatcttacc
    10681 gctgttgaga tccagttcga tgtaacccac tcgtgcaccc aactgatctt cagcatcttt
    10741 tactttcacc agcgtttctg ggtgagcaaa aacgggaagg caaaatgccg caaaaaaggg
    10801 aataagggcg acacggaaat gttgaatact catactcttc ctttttcaat attattgaag
    10861 catttatcag ggttattgtc tcatgagcgg atacatattt gaatgtattt agaaaaataa
    10921 acaaataggg gttccgcgca catttccccg aaaagtgcca c
//
