LOCUS       sequence-313846-       11659 bp    DNA     circular SYN 15-DEC-2025
DEFINITION  For cloning sgRNA to knockout a specific gene in piggybac plasmid.
ACCESSION   .
VERSION     .
KEYWORDS    .
SOURCE      synthetic DNA construct
  ORGANISM  synthetic DNA construct
REFERENCE   1  (bases 1 to 11659)
  AUTHORS   Jiang Y, Hoenisch RC, Chang Y, Bao X, Cameron CE, Lian XL
  TITLE     Robust genome and RNA editing via CRISPR nucleases in PiggyBac 
            systems.
  JOURNAL   Bioact Mater. 2022 Feb 7;14:313-320. doi: 
            10.1016/j.bioactmat.2022.01.046. eCollection 2022 Aug.
  PUBMED    35386818
REFERENCE   2  (bases 1 to 11659)
  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: "Bioact 
            Mater."; date: "2022-02-7"; volume: "14:313-320. doi"; pages: " 
            10.1016/j.bioactmat.2022.01.046. eCollection 2022 Aug"
COMMENT     Sequence Label: PB-CRISPR
FEATURES             Location/Qualifiers
     source          1..11659
                     /mol_type="other DNA"
                     /organism="synthetic DNA construct"
     rep_origin      complement(83..538)
                     /direction=LEFT
                     /label=f1 ori
                     /note="f1 bacteriophage origin of replication; arrow 
                     indicates direction of (+) strand synthesis"
     primer_bind     complement(220..241)
                     /label=F1ori-F
                     /note="F1 origin, forward primer"
     primer_bind     432..451
                     /label=F1ori-R
                     /note="F1 origin, reverse primer"
     primer_bind     665..687
                     /label=M13/pUC Forward
                     /note="In lacZ gene"
     primer_bind     679..696
                     /label=M13 Forward
                     /note="In lacZ gene. Also called M13-F20 or M13 (-21) 
                     Forward"
     primer_bind     680..696
                     /label=M13 fwd
                     /note="common sequencing primer, one of multiple similar 
                     variants"
     primer_bind     706..725
                     /label=T7
                     /note="T7 promoter, forward primer"
     promoter        706..724
                     /label=T7 promoter
                     /note="promoter for bacteriophage T7 RNA polymerase"
     repeat_region   754..788
                     /label=piggyBac left (5') inverted repeat
                     /note="piggyBac transposon-specific inverted terminal 
                     repeat sequence (ITR)"
     promoter        1078..1326
                     /label=U6 promoter
                     /note="RNA polymerase III promoter for human U6 snRNA 
                     (Domitrovich & Kunkel, 2003)"
     primer_bind     1078..1098
                     /label=hU6-F
                     /note="Human U6 promoter, forward primer"
     primer_bind     1249..1268
                     /label=LKO.1 5'
                     /note="Human U6 promoter, forward primer"
     misc_RNA        3208..3283
                     /label=gRNA scaffold
                     /note="guide RNA scaffold for the Streptococcus pyogenes 
                     CRISPR/Cas9 system"
     misc_feature    3331..3448
                     /label=cPPT/CTS
                     /note="central polypurine tract and central termination 
                     sequence of HIV-1"
     promoter        3517..3728
                     /label=EF-1-alpha core promoter
                     /note="core promoter for human elongation factor 
                     EF-1-alpha"
     regulatory      3748..3757
                     /label=Kozak sequence
                     /note="vertebrate consensus sequence for strong initiation 
                     of translation (Kozak, 1987)"
                     /regulatory_class="other"
     CDS             3757..3780
                     /codon_start=1
                     /product="FLAG(R) epitope tag, followed by an enterokinase 
                     cleavage site"
                     /label=FLAG
                     /translation="DYKDDDDK"
     CDS             3787..3807
                     /codon_start=1
                     /product="nuclear localization signal of SV40 (simian virus
                     40) large T antigen"
                     /label=SV40 NLS
                     /translation="PKKKRKV"
     CDS             3832..7932
                     /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             3832..7932
                     /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             7933..7980
                     /codon_start=1
                     /product="bipartite nuclear localization signal from 
                     nucleoplasmin"
                     /label=nucleoplasmin NLS
                     /translation="KRPAATKKAGQAKKKK"
     CDS             7996..8052
                     /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             8053..8652
                     /codon_start=1
                     /gene="pac from Streptomyces alboniger"
                     /product="puromycin N-acetyltransferase"
                     /label=PuroR
                     /note="confers resistance to puromycin"
                     /translation="MTEYKPTVRLATRDDVPRAVRTLAAAFADYPATRHTVDPDRHIER
                     VTELQELFLTRVGLDIGKVWVADDGAAVAVWTTPESVEAGAVFAEIGPRMAELSGSRLA
                     AQQQMEGLLAPHRPKEPAWFLATVGVSPDHQGKGLGSAVVLPGVEAAERAGVPAFLETS
                     APRNLPFYERLGFTVTADVEVPEGPRTWCMTRKPGA"
     primer_bind     complement(8053..8072)
                     /label=Puro-R
                     /note="Puromycin resistance gene, reverse primer. Also 
                     called puro-variant-R"
     primer_bind     8549..8569
                     /label=Puro-F
                     /note="Puromycin resistance gene, forward primer"
     misc_feature    8668..9256
                     /label=WPRE
                     /note="woodchuck hepatitis virus posttranscriptional 
                     regulatory element"
     primer_bind     complement(8721..8741)
                     /label=WPRE-R
                     /note="WPRE, reverse primer"
     CDS             complement(9139..9150)
                     /codon_start=1
                     /product="Factor Xa recognition and cleavage site"
                     /label=Factor Xa site
                     /translation="IEGR"
     repeat_region   9438..9500
                     /label=piggyBac right (3') inverted repeat
                     /note="piggyBac transposon-specific inverted terminal 
                     repeat sequence (ITR)"
     primer_bind     complement(9551..9571)
                     /label=T3
                     /note="T3 promoter, forward primer"
     promoter        complement(9551..9569)
                     /label=T3 promoter
                     /note="promoter for bacteriophage T3 RNA polymerase"
     primer_bind     complement(9590..9606)
                     /label=M13 rev
                     /note="common sequencing primer, one of multiple similar 
                     variants"
     primer_bind     complement(9590..9606)
                     /label=M13 Reverse
                     /note="In lacZ gene. Also called M13-rev"
     primer_bind     complement(9603..9625)
                     /label=M13/pUC Reverse
                     /note="In lacZ gene"
     protein_bind    9614..9630
                     /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(9638..9644,9645..9662,9663..9668))
                     /label=lac promoter
                     /note="promoter for the E. coli lac operon"
     protein_bind    9683..9704
                     /label=CAP binding site
                     /bound_moiety="E. coli catabolite activator protein"
                     /note="CAP binding activates transcription in the presence 
                     of cAMP."
     primer_bind     complement(9821..9838)
                     /label=L4440
                     /note="L4440 vector, forward primer"
     rep_origin      complement(9992..10580)
                     /direction=LEFT
                     /label=ori
                     /note="high-copy-number ColE1/pMB1/pBR322/pUC origin of 
                     replication"
     primer_bind     complement(10072..10091)
                     /label=pBR322ori-F
                     /note="pBR322 origin, forward primer"
     CDS             complement(join(10751..11542,11543..11611))
                     /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     11374..11393
                     /label=Amp-R
                     /note="Ampicillin resistance gene, reverse primer"
     promoter        complement(join(11612..11659,1..57))
                     /gene="bla"
                     /label=AmpR promoter
ORIGIN
        1 catgagcgga tacatatttg aatgtattta gaaaaataaa caaatagggg ttccgcgcac
       61 atttccccga aaagtgccac ctaaattgta agcgttaata ttttgttaaa attcgcgtta
      121 aatttttgtt aaatcagctc attttttaac caataggccg aaatcggcaa aatcccttat
      181 aaatcaaaag aatagaccga gatagggttg agtgttgttc cagtttggaa caagagtcca
      241 ctattaaaga acgtggactc caacgtcaaa gggcgaaaaa ccgtctatca gggcgatggc
      301 ccactacgtg aaccatcacc ctaatcaagt tttttggggt cgaggtgccg taaagcacta
      361 aatcggaacc ctaaagggag cccccgattt agagcttgac ggggaaagcc ggcgaacgtg
      421 gcgagaaagg aagggaagaa agcgaaagga gcgggcgcta gggcgctggc aagtgtagcg
      481 gtcacgctgc gcgtaaccac cacacccgcc gcgcttaatg cgccgctaca gggcgcgtcc
      541 cattcgccat tcaggctgcg caactgttgg gaagggcgat cggtgcgggc ctcttcgcta
      601 ttacgccagc tggcgaaagg gggatgtgct gcaaggcgat taagttgggt aacgccaggg
      661 ttttcccagt cacgacgttg taaaacgacg gccagtgagc gcgcgtaata cgactcacta
      721 tagggcgaat tggggcgcgc cattctagat taaccctaga aagatagtct gcgtaaaatt
      781 gacgcatgca ttcttgaaat attgctctct ctttctaaat agcgcgaatc cgtcgctgtg
      841 catttaggac atctcagtcg ccgcttggag ctcccgtgag gcgtgcttgt caatgcggta
      901 agtgtcactg attttgaact ataacgaccg cgtgagtcaa aatgacgcat gattatcttt
      961 tacgtgactt ttaagattta actcatacga taattatatt gttatttcat gttctactta
     1021 cgtgataact tattatatat atattttctt gttatagata tcaactagaa tgctagcgag
     1081 ggcctatttc ccatgattcc ttcatatttg catatacgat acaaggctgt tagagagata
     1141 attagaatta atttgactgt aaacacaaag atattagtac aaaatacgtg acgtagaaag
     1201 taataatttc ttgggtagtt tgcagtttta aaattatgtt ttaaaatgga ctatcatatg
     1261 cttaccgtaa cttgaaagta tttcgatttc ttggctttat atatcttgtg gaaaggacga
     1321 aacaccggag acggttgtaa atgagcacac aaaatacaca tgctaaaata ttatattcta
     1381 tgacctttat aaaatcaacc aaaatcttct ttttaataac tttagtatca ataattagaa
     1441 tttttatgtt cctttttgca aacttttaat aaaaatgagc aaaataaaaa aacgctagtt
     1501 ttagtaactc gcgttgtttt cttcaccttt aataatagct actccaccac ttgttcctaa
     1561 gcggtcagct cctgcttcaa tcattttttg agcatcttca aatgttctaa ctccaccagc
     1621 tgctttaact aaagcattgt ctttaacaac tgacttcatt agtttaacat cttcaaatgt
     1681 tgcacctgat tttgaaaatc ctgttgatgt tttaacaaat tctaatccag cttcaacagc
     1741 tatttcacaa gctttcatga tttcttcttt tgttaataaa caattttcca taatacattt
     1801 aacaacatgt gatccagctg ctttttttac agctttcatg tcttctaaaa ctaattcata
     1861 atttttgtct tttaatgcac caatatttaa taccatatca atttctgttg caccatcttt
     1921 aattgcttca gaaacttcga atgcttttgt agctgttgtg catgcaccta gaggaaaacc
     1981 tacaacattt gttattccta catttgtgcc ttttaataat tctttacaat agcttgttca
     2041 atatgaatta acacaaactg ttgcaaaatc aaattcaatt gcttcatcac ataattgttt
     2101 aatttcagct ttcgtagcat cttgttttaa taatgtgtga tctatatatt tgtttagttt
     2161 cattttttct cctatatatt catttttaat tttaattctt taataatttc gtctacttta
     2221 actttagcgt tttgaacaga ttcaccaaca cctataaaat aaatttttag tttaggttca
     2281 gttccacttg ggcgaacagc aaatcatgac ttatcttcta aataaaattt tagtaagtct
     2341 tgtcctggca tattatacat tccatcgatg tagtcttcaa cattaacaac tttaagtcca
     2401 gcaatttgag ttaagggtgt tgctctcaat gatttcatta atggttcaat ttttaatttc
     2461 ttttcttctg gtttaaaatt caagtttaaa gtgaaagtgt aatatgcacc catttcttta
     2521 aataaatctt ctaaatagtc tactaatgtt ttattttgtt ttttataaaa tcaagcagcc
     2581 tctgctatta atatagaagc ttgtattcca tctttatctc tagctgagtc atcaattaca
     2641 tatccataac tttcttcata agcaaaaaca aaatttaatc cgttatcttc ttctttagca
     2701 atttctctac ccattcattt aaatccagtt aaagttttta caatattaac tccatatttt
     2761 tcatgagcga ttctatcacc caaatcactt gttacaaaac ttgaatatag agccggattt
     2821 tttggaatgc tatttaagcg ttttagattt gataattttc aatcaattaa aattggtcct
     2881 gtttgatttc catctaatct tacaaaatga ccatcatgtt ttattgccat tccaaatctg
     2941 tcagcatctg ggtcattcat aataataata tctgcatcat gtttaatacc atattcaagc
     3001 ggtatttttc atgcaggatc aaattctgga tttggattta caacattttt aaatgtttca
     3061 tcttcaaatg catgctcttc aacctcaata acgttatatc ctgattcacg taatattttt
     3121 ggggtaaatt tagttcctgt tccattaact gcgctaaaaa taatttttaa atctttttta
     3181 gcttcttgct cttttttgta cgtctctgtt ttagagctag aaatagcaag ttaaaataag
     3241 gctagtccgt tatcaacttg aaaaagtggc accgagtcgg tgcttttttg aattctagat
     3301 cttgagacaa atggcagtat tcatccacaa ttttaaaaga aaagggggga ttggggggta
     3361 cagtgcaggg gaaagaatag tagacataat agcaacagac atacaaacta aagaattaca
     3421 aaaacaaatt acaaaaattc aaaattttcg ggtttattac agggacagca gagatccact
     3481 ttggcgccgg ctcgagtggc tccggtgccc gtcagtgggc agagcgcaca tcgcccacag
     3541 tccccgagaa gttgggggga ggggtcggca attgaaccgg tgcctagaga aggtggcgcg
     3601 gggtaaactg ggaaagtgat gtcgtgtact ggctccgcct ttttcccgag ggtgggggag
     3661 aaccgtatat aagtgcagta gtcgccgtga acgttctttt tcgcaacggg tttgccgcca
     3721 gaacacaggt gtcgtgacgc gggatccgcc accatggatt acaaagacga tgacgataag
     3781 atggccccaa agaagaagcg gaaggtcggt atccacggag tcccagcagc cgacaagaag
     3841 tacagcatcg gcctggacat cggcaccaac tctgtgggct gggccgtgat caccgacgag
     3901 tacaaggtgc ccagcaagaa attcaaggtg ctgggcaaca ccgaccggca cagcatcaag
     3961 aagaacctga tcggagccct gctgttcgac agcggcgaaa cagccgaggc cacccggctg
     4021 aagagaaccg ccagaagaag atacaccaga cggaagaacc ggatctgcta tctgcaagag
     4081 atcttcagca acgagatggc caaggtggac gacagcttct tccacagact ggaagagtcc
     4141 ttcctggtgg aagaggataa gaagcacgag cggcacccca tcttcggcaa catcgtggac
     4201 gaggtggcct accacgagaa gtaccccacc atctaccacc tgagaaagaa actggtggac
     4261 agcaccgaca aggccgacct gcggctgatc tatctggccc tggcccacat gatcaagttc
     4321 cggggccact tcctgatcga gggcgacctg aaccccgaca acagcgacgt ggacaagctg
     4381 ttcatccagc tggtgcagac ctacaaccag ctgttcgagg aaaaccccat caacgccagc
     4441 ggcgtggacg ccaaggccat cctgtctgcc agactgagca agagcagacg gctggaaaat
     4501 ctgatcgccc agctgcccgg cgagaagaag aatggcctgt tcggaaacct gattgccctg
     4561 agcctgggcc tgacccccaa cttcaagagc aacttcgacc tggccgagga tgccaaactg
     4621 cagctgagca aggacaccta cgacgacgac ctggacaacc tgctggccca gatcggcgac
     4681 cagtacgccg acctgtttct ggccgccaag aacctgtccg acgccatcct gctgagcgac
     4741 atcctgagag tgaacaccga gatcaccaag gcccccctga gcgcctctat gatcaagaga
     4801 tacgacgagc accaccagga cctgaccctg ctgaaagctc tcgtgcggca gcagctgcct
     4861 gagaagtaca aagagatttt cttcgaccag agcaagaacg gctacgccgg ctacattgac
     4921 ggcggagcca gccaggaaga gttctacaag ttcatcaagc ccatcctgga aaagatggac
     4981 ggcaccgagg aactgctcgt gaagctgaac agagaggacc tgctgcggaa gcagcggacc
     5041 ttcgacaacg gcagcatccc ccaccagatc cacctgggag agctgcacgc cattctgcgg
     5101 cggcaggaag atttttaccc attcctgaag gacaaccggg aaaagatcga gaagatcctg
     5161 accttccgca tcccctacta cgtgggccct ctggccaggg gaaacagcag attcgcctgg
     5221 atgaccagaa agagcgagga aaccatcacc ccctggaact tcgaggaagt ggtggacaag
     5281 ggcgcttccg cccagagctt catcgagcgg atgaccaact tcgataagaa cctgcccaac
     5341 gagaaggtgc tgcccaagca cagcctgctg tacgagtact tcaccgtgta taacgagctg
     5401 accaaagtga aatacgtgac cgagggaatg agaaagcccg ccttcctgag cggcgagcag
     5461 aaaaaggcca tcgtggacct gctgttcaag accaaccgga aagtgaccgt gaagcagctg
     5521 aaagaggact acttcaagaa aatcgagtgc ttcgactccg tggaaatctc cggcgtggaa
     5581 gatcggttca acgcctccct gggcacatac cacgatctgc tgaaaattat caaggacaag
     5641 gacttcctgg acaatgagga aaacgaggac attctggaag atatcgtgct gaccctgaca
     5701 ctgtttgagg acagagagat gatcgaggaa cggctgaaaa cctatgccca cctgttcgac
     5761 gacaaagtga tgaagcagct gaagcggcgg agatacaccg gctggggcag gctgagccgg
     5821 aagctgatca acggcatccg ggacaagcag tccggcaaga caatcctgga tttcctgaag
     5881 tccgacggct tcgccaacag aaacttcatg cagctgatcc acgacgacag cctgaccttt
     5941 aaagaggaca tccagaaagc ccaggtgtcc ggccagggcg atagcctgca cgagcacatt
     6001 gccaatctgg ccggcagccc cgccattaag aagggcatcc tgcagacagt gaaggtggtg
     6061 gacgagctcg tgaaagtgat gggccggcac aagcccgaga acatcgtgat cgaaatggcc
     6121 agagagaacc agaccaccca gaagggacag aagaacagcc gcgagagaat gaagcggatc
     6181 gaagagggca tcaaagagct gggcagccag atcctgaaag aacaccccgt ggaaaacacc
     6241 cagctgcaga acgagaagct gtacctgtac tacctgcaga atgggcggga tatgtacgtg
     6301 gaccaggaac tggacatcaa ccggctgtcc gactacgatg tggaccatat cgtgcctcag
     6361 agctttctga aggacgactc catcgacaac aaggtgctga ccagaagcga caagaaccgg
     6421 ggcaagagcg acaacgtgcc ctccgaagag gtcgtgaaga agatgaagaa ctactggcgg
     6481 cagctgctga acgccaagct gattacccag agaaagttcg acaatctgac caaggccgag
     6541 agaggcggcc tgagcgaact ggataaggcc ggcttcatca agagacagct ggtggaaacc
     6601 cggcagatca caaagcacgt ggcacagatc ctggactccc ggatgaacac taagtacgac
     6661 gagaatgaca agctgatccg ggaagtgaaa gtgatcaccc tgaagtccaa gctggtgtcc
     6721 gatttccgga aggatttcca gttttacaaa gtgcgcgaga tcaacaacta ccaccacgcc
     6781 cacgacgcct acctgaacgc cgtcgtggga accgccctga tcaaaaagta ccctaagctg
     6841 gaaagcgagt tcgtgtacgg cgactacaag gtgtacgacg tgcggaagat gatcgccaag
     6901 agcgagcagg aaatcggcaa ggctaccgcc aagtacttct tctacagcaa catcatgaac
     6961 tttttcaaga ccgagattac cctggccaac ggcgagatcc ggaagcggcc tctgatcgag
     7021 acaaacggcg aaaccgggga gatcgtgtgg gataagggcc gggattttgc caccgtgcgg
     7081 aaagtgctga gcatgcccca agtgaatatc gtgaaaaaga ccgaggtgca gacaggcggc
     7141 ttcagcaaag agtctatcct gcccaagagg aacagcgata agctgatcgc cagaaagaag
     7201 gactgggacc ctaagaagta cggcggcttc gacagcccca ccgtggccta ttctgtgctg
     7261 gtggtggcca aagtggaaaa gggcaagtcc aagaaactga agagtgtgaa agagctgctg
     7321 gggatcacca tcatggaaag aagcagcttc gagaagaatc ccatcgactt tctggaagcc
     7381 aagggctaca aagaagtgaa aaaggacctg atcatcaagc tgcctaagta ctccctgttc
     7441 gagctggaaa acggccggaa gagaatgctg gcctctgccg gcgaactgca gaagggaaac
     7501 gaactggccc tgccctccaa atatgtgaac ttcctgtacc tggccagcca ctatgagaag
     7561 ctgaagggct cccccgagga taatgagcag aaacagctgt ttgtggaaca gcacaagcac
     7621 tacctggacg agatcatcga gcagatcagc gagttctcca agagagtgat cctggccgac
     7681 gctaatctgg acaaagtgct gtccgcctac aacaagcacc gggataagcc catcagagag
     7741 caggccgaga atatcatcca cctgtttacc ctgaccaatc tgggagcccc tgccgccttc
     7801 aagtactttg acaccaccat cgaccggaag aggtacacca gcaccaaaga ggtgctggac
     7861 gccaccctga tccaccagag catcaccggc ctgtacgaga cacggatcga cctgtctcag
     7921 ctgggaggcg acaagcgtcc tgctgctact aagaaagctg gtcaagctaa gaaaaagaaa
     7981 gctagcggca gcggcgccac caacttcagc ctgctgaagc aggccggcga cgtggaggag
     8041 aaccccggcc ccatgaccga gtacaagccc acggtgcgcc tcgccacccg cgacgacgtc
     8101 cccagggccg tacgcaccct cgccgccgcg ttcgccgact accccgccac gcgccacacc
     8161 gtcgatccgg accgccacat cgagcgggtc accgagctgc aagaactctt cctcacgcgc
     8221 gtcgggctcg acatcggcaa ggtgtgggtc gcggacgacg gcgccgcggt ggcggtctgg
     8281 accacgccgg agagcgtcga agcgggggcg gtgttcgccg agatcggccc gcgcatggcc
     8341 gagttgagcg gttcccggct ggccgcgcag caacagatgg aaggcctcct ggcgccgcac
     8401 cggcccaagg agcccgcgtg gttcctggcc accgtcggag tctcgcccga ccaccagggc
     8461 aagggtctgg gcagcgccgt cgtgctcccc ggagtggagg cggccgagcg cgccggggtg
     8521 cccgccttcc tggagacctc cgcgccccgc aacctcccct tctacgagcg gctcggcttc
     8581 accgtcaccg ccgacgtcga ggtgcccgaa ggaccgcgca cctggtgcat gacccgcaag
     8641 cccggtgcct gaacgcgtta agtcgacaat caacctctgg attacaaaat ttgtgaaaga
     8701 ttgactggta ttcttaacta tgttgctcct tttacgctat gtggatacgc tgctttaatg
     8761 cctttgtatc atgctattgc ttcccgtatg gctttcattt tctcctcctt gtataaatcc
     8821 tggttgctgt ctctttatga ggagttgtgg cccgttgtca ggcaacgtgg cgtggtgtgc
     8881 actgtgtttg ctgacgcaac ccccactggt tggggcattg ccaccacctg tcagctcctt
     8941 tccgggactt tcgctttccc cctccctatt gccacggcgg aactcatcgc cgcctgcctt
     9001 gcccgctgct ggacaggggc tcggctgttg ggcactgaca attccgtggt gttgtcgggg
     9061 aaatcatcgt cctttccttg gctgctcgcc tgtgttgcca cctggattct gcgcgggacg
     9121 tccttctgct acgtcccttc ggccctcaat ccagcggacc ttccttcccg cggcctgctg
     9181 ccggctctgc ggcctcttcc gcgtcttcgc cttcgccctc agacgagtcg gatctccctt
     9241 tgggccgcct ccccgcacta gttaaaagtt ttgttacttt atagaagaaa ttttgagttt
     9301 ttgttttttt ttaataaata aataaacata aataaattgt ttgttgaatt tattattagt
     9361 atgtaagtgt aaatataata aaacttaata tctattcaaa ttaataaata aacctcgata
     9421 tacagaccga taaaacacat gcgtcaattt tacgcatgat tatctttaac gtacgtcaca
     9481 atatgattat ctttctaggg ttaatctagt atacgcgtat gcggccgctt aattaatcca
     9541 gcttttgttc cctttagtga gggttaattg cgcgcttggc gtaatcatgg tcatagctgt
     9601 ttcctgtgtg aaattgttat ccgctcacaa ttccacacaa catacgagcc ggaagcataa
     9661 agtgtaaagc ctggggtgcc taatgagtga gctaactcac attaattgcg ttgcgctcac
     9721 tgcccgcttt ccagtcggga aacctgtcgt gccagctgca ttaatgaatc ggccaacgcg
     9781 cggggagagg cggtttgcgt attgggcgct cttccgcttc ctcgctcact gactcgctgc
     9841 gctcggtcgt tcggctgcgg cgagcggtat cagctcactc aaaggcggta atacggttat
     9901 ccacagaatc aggggataac gcaggaaaga acatgtgagc aaaaggccag caaaaggcca
     9961 ggaaccgtaa aaaggccgcg ttgctggcgt ttttccatag gctccgcccc cctgacgagc
    10021 atcacaaaaa tcgacgctca agtcagaggt ggcgaaaccc gacaggacta taaagatacc
    10081 aggcgtttcc ccctggaagc tccctcgtgc gctctcctgt tccgaccctg ccgcttaccg
    10141 gatacctgtc cgcctttctc ccttcgggaa gcgtggcgct ttctcatagc tcacgctgta
    10201 ggtatctcag ttcggtgtag gtcgttcgct ccaagctggg ctgtgtgcac gaaccccccg
    10261 ttcagcccga ccgctgcgcc ttatccggta actatcgtct tgagtccaac ccggtaagac
    10321 acgacttatc gccactggca gcagccactg gtaacaggat tagcagagcg aggtatgtag
    10381 gcggtgctac agagttcttg aagtggtggc ctaactacgg ctacactaga agaacagtat
    10441 ttggtatctg cgctctgctg aagccagtta ccttcggaaa aagagttggt agctcttgat
    10501 ccggcaaaca aaccaccgct ggtagcggtg gtttttttgt ttgcaagcag cagattacgc
    10561 gcagaaaaaa aggatctcaa gaagatcctt tgatcttttc tacggggtct gacgctcagt
    10621 ggaacgaaaa ctcacgttaa gggattttgg tcatgagatt atcaaaaagg atcttcacct
    10681 agatcctttt aaattaaaaa tgaagtttta aatcaatcta aagtatatat gagtaaactt
    10741 ggtctgacag ttaccaatgc ttaatcagtg aggcacctat ctcagcgatc tgtctatttc
    10801 gttcatccat agttgcctga ctccccgtcg tgtagataac tacgatacgg gagggcttac
    10861 catctggccc cagtgctgca atgataccgc gagacccacg ctcaccggct ccagatttat
    10921 cagcaataaa ccagccagcc ggaagggccg agcgcagaag tggtcctgca actttatccg
    10981 cctccatcca gtctattaat tgttgccggg aagctagagt aagtagttcg ccagttaata
    11041 gtttgcgcaa cgttgttgcc attgctacag gcatcgtggt gtcacgctcg tcgtttggta
    11101 tggcttcatt cagctccggt tcccaacgat caaggcgagt tacatgatcc cccatgttgt
    11161 gcaaaaaagc ggttagctcc ttcggtcctc cgatcgttgt cagaagtaag ttggccgcag
    11221 tgttatcact catggttatg gcagcactgc ataattctct tactgtcatg ccatccgtaa
    11281 gatgcttttc tgtgactggt gagtactcaa ccaagtcatt ctgagaatag tgtatgcggc
    11341 gaccgagttg ctcttgcccg gcgtcaatac gggataatac cgcgccacat agcagaactt
    11401 taaaagtgct catcattgga aaacgttctt cggggcgaaa actctcaagg atcttaccgc
    11461 tgttgagatc cagttcgatg taacccactc gtgcacccaa ctgatcttca gcatctttta
    11521 ctttcaccag cgtttctggg tgagcaaaaa caggaaggca aaatgccgca aaaaagggaa
    11581 taagggcgac acggaaatgt tgaatactca tactcttcct ttttcaatat tattgaagca
    11641 tttatcaggg ttattgtct
//
