LOCUS       sequence-329724-       11025 bp    DNA     circular SYN 15-DEC-2025
DEFINITION  PiggyBac transposon vector constitutively driving PuroR-eGFP and 
            conditionally expressing S.aureus Cas9 and mCherry .
ACCESSION   .
VERSION     .
KEYWORDS    .
SOURCE      synthetic DNA construct
  ORGANISM  synthetic DNA construct
REFERENCE   1  (bases 1 to 11025)
  AUTHORS   Biegler MT, Fedrigo O, Collier P, Mountcastle J, Haase B, Tilgner 
            HU, Jarvis ED
  TITLE     Induction of an immortalized songbird cell line allows for gene 
            characterization and knockout by CRISPR-Cas9.
  JOURNAL   Sci Rep. 2022 Mar 14;12(1):4369. doi: 10.1038/s41598-022-07434-7.
  PUBMED    35288582
REFERENCE   2  (bases 1 to 11025)
  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: "Sci Rep."; 
            date: "2022-03-14"; volume: "12(1):4369. doi"; pages: " 
            10.1038/s41598-022-07434-7"
COMMENT     Sequence Label: 
            pMB952:PB-CAG-PuroR-nGFP-FLEx-SaCas9-P2A-mCherry-WPRE
FEATURES             Location/Qualifiers
     source          1..11025
                     /mol_type="other DNA"
                     /organism="synthetic DNA construct"
     repeat_region   111..173
                     /label=piggyBac right (3') inverted repeat
                     /note="piggyBac transposon-specific inverted terminal 
                     repeat sequence (ITR)"
     enhancer        245..548
                     /label=CMV enhancer
                     /note="human cytomegalovirus immediate early enhancer"
     intron          815..1831
                     /label=chimeric intron
                     /note="chimera between introns from chicken beta-actin and 
                     rabbit beta-globin"
     primer_bind     1755..1777
                     /label=pCAGGS-5
                     /note="Chimeric intron in CAG promoter, forward primer"
     primer_bind     1839..1858
                     /label=pCAG-F
                     /note="Rabbit beta-globin intron, for pCAG plasmids, 
                     forward primer"
     protein_bind    1881..1914
                     /label=loxP
                     /bound_moiety="Cre recombinase"
                     /note="Cre-mediated recombination occurs in the 8-bp core 
                     sequence (ATGTATGC) (Shaw et al., 2021)."
     protein_bind    1915..1948
                     /label=FRT (minimal)
                     /bound_moiety="FLP recombinase from the Saccharomyces 
                     cerevisiae 2u plasmid"
                     /note="supports FLP-mediated excision but not integration 
                     (Turan and Bode, 2011)"
     CDS             1949..2548
                     /codon_start=1
                     /gene="pac from Streptomyces alboniger"
                     /product="puromycin N-acetyltransferase"
                     /label=PuroR
                     /note="confers resistance to puromycin"
                     /translation="MATEYKPTVRLATRDDVPRAVRTLAAAFADYPATRHTVDPDRHIE
                     RVTELQELFLTRVGLDIGKVWVADDGAAVAVWTTPESVEAGAVFAEIGPRMAELSGSRL
                     AAQQQMEGLLAPHRPKEPAWFLATVGVSPDHQGKGLGSAVVLPGVEAAERAGVPAFLET
                     SAPRNLPFYERLGFTVTADVEVPEGPRTWCMTRKPGA"
     primer_bind     2448..2468
                     /label=Puro-F
                     /note="Puromycin resistance gene, forward primer"
     CDS             2564..2620
                     /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             2621..2641
                     /codon_start=1
                     /product="nuclear localization signal of SV40 (simian virus
                     40) large T antigen"
                     /label=SV40 NLS
                     /translation="PKKKRKV"
     CDS             2645..2665
                     /codon_start=1
                     /product="nuclear localization signal of SV40 (simian virus
                     40) large T antigen"
                     /label=SV40 NLS
                     /translation="PKKKRKV"
     CDS             2669..2689
                     /codon_start=1
                     /product="nuclear localization signal of SV40 (simian virus
                     40) large T antigen"
                     /label=SV40 NLS
                     /translation="PKKKRKV"
     regulatory      2705..2714
                     /label=Kozak sequence
                     /note="vertebrate consensus sequence for strong initiation 
                     of translation (Kozak, 1987)"
                     /regulatory_class="other"
     CDS             join(2711..2713,2714..2716,2717..3430)
                     /codon_start=1
                     /product="the original enhanced GFP (Yang et al., 1996)"
                     /label=EGFP
                     /note="mammalian codon-optimized"
                     /translation="MVSKGEELFTGVVPILVELDGDVNGHKFSVSGEGEGDATYGKLTL
                     KFICTTGKLPVPWPTLVTTLTYGVQCFSRYPDHMKQHDFFKSAMPEGYVQERTIFFKDD
                     GNYKTRAEVKFEGDTLVNRIELKGIDFKEDGNILGHKLEYNYNSHNVYIMADKQKNGIK
                     VNFKIRHNIEDGSVQLADHYQQNTPIGDGPVLLPDNHYLSTQSALSKDPNEKRDHMVLL
                     EFVTAAGITLGMDELYK"
     CDS             join(2711..2713,2714..2716,2717..3427)
                     /codon_start=1
                     /product="enhanced GFP"
                     /label=EGFP
                     /translation="MVSKGEELFTGVVPILVELDGDVNGHKFSVSGEGEGDATYGKLTL
                     KFICTTGKLPVPWPTLVTTLTYGVQCFSRYPDHMKQHDFFKSAMPEGYVQERTIFFKDD
                     GNYKTRAEVKFEGDTLVNRIELKGIDFKEDGNILGHKLEYNYNSHNVYIMADKQKNGIK
                     VNFKIRHNIEDGSVQLADHYQQNTPIGDGPVLLPDNHYLSTQSALSKDPNEKRDHMVLL
                     EFVTAAGITLGMDELYK"
     primer_bind     complement(2756..2777)
                     /label=EGFP-N
                     /note="EGFP, reverse primer"
     primer_bind     complement(3017..3036)
                     /label=EXFP-R
                     /note="For distinguishing EGFP variants, reverse primer"
     primer_bind     3364..3385
                     /label=EGFP-C
                     /note="EGFP, forward primer"
     protein_bind    3434..3467
                     /label=FRT (minimal)
                     /bound_moiety="FLP recombinase from the Saccharomyces 
                     cerevisiae 2u plasmid"
                     /note="supports FLP-mediated excision but not integration 
                     (Turan and Bode, 2011)"
     polyA_signal    3468..3516
                     /note="synthetic polyadenylation signal"
     protein_bind    3517..3550
                     /label=lox2272
                     /bound_moiety="Cre recombinase"
                     /note="Cre-mediated recombination occurs in the 8-bp core 
                     sequence (AAGTATCC) (Shaw et al., 2021). lox2272 sites are 
                     compatible with each other, but incompatible with loxP or 
                     loxN sites (Lee and Saito, 1988)."
     CDS             complement(3558..4265)
                     /codon_start=1
                     /product="monomeric derivative of DsRed fluorescent protein
                     (Shaner et al., 2004)"
                     /label=mCherry
                     /note="mammalian codon-optimized"
                     /translation="VSKGEEDNMAIIKEFMRFKVHMEGSVNGHEFEIEGEGEGRPYEGT
                     QTAKLKVTKGGPLPFAWDILSPQFMYGSKAYVKHPADIPDYLKLSFPEGFKWERVMNFE
                     DGGVVTVTQDSSLQDGEFIYKVKLRGTNFPSDGPVMQKKTMGWEASSERMYPEDGALKG
                     EIKQRLKLKDGGHYDAEVKTTYKAKKPVQLPGAYNVNIKLDITSHNEDYTIVEQYERAE
                     GRHSTGGMDELYK"
     primer_bind     complement(3836..3855)
                     /label=mCherry-F
                     /note="mCherry, forward primer"
     primer_bind     4105..4123
                     /label=mCherry-R
                     /note="mCherry, reverse primer"
     CDS             complement(4272..4328)
                     /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             complement(4341..4367)
                     /codon_start=1
                     /product="HA (human influenza hemagglutinin) epitope tag"
                     /label=HA
                     /translation="YPYDVPDYA"
     CDS             complement(4368..4394)
                     /codon_start=1
                     /product="HA (human influenza hemagglutinin) epitope tag"
                     /label=HA
                     /translation="YPYDVPDYA"
     CDS             complement(4401..4448)
                     /codon_start=1
                     /product="bipartite nuclear localization signal from 
                     nucleoplasmin"
                     /label=nucleoplasmin NLS
                     /translation="KRPAATKKAGQAKKKK"
     CDS             complement(4449..7604)
                     /codon_start=1
                     /product="Cas9 endonuclease from the Staphylococcus aureus 
                     Type II CRISPR/Cas system"
                     /label=SaCas9
                     /note="generates RNA-guided double strand breaks in DNA"
                     /translation="KRNYILGLDIGITSVGYGIIDYETRDVIDAGVRLFKEANVENNEG
                     RRSKRGARRLKRRRRHRIQRVKKLLFDYNLLTDHSELSGINPYEARVKGLSQKLSEEEF
                     SAALLHLAKRRGVHNVNEVEEDTGNELSTKEQISRNSKALEEKYVAELQLERLKKDGEV
                     RGSINRFKTSDYVKEAKQLLKVQKAYHQLDQSFIDTYIDLLETRRTYYEGPGEGSPFGW
                     KDIKEWYEMLMGHCTYFPEELRSVKYAYNADLYNALNDLNNLVITRDENEKLEYYEKFQ
                     IIENVFKQKKKPTLKQIAKEILVNEEDIKGYRVTSTGKPEFTNLKVYHDIKDITARKEI
                     IENAELLDQIAKILTIYQSSEDIQEELTNLNSELTQEEIEQISNLKGYTGTHNLSLKAI
                     NLILDELWHTNDNQIAIFNRLKLVPKKVDLSQQKEIPTTLVDDFILSPVVKRSFIQSIK
                     VINAIIKKYGLPNDIIIELAREKNSKDAQKMINEMQKRNRQTNERIEEIIRTTGKENAK
                     YLIEKIKLHDMQEGKCLYSLEAIPLEDLLNNPFNYEVDHIIPRSVSFDNSFNNKVLVKQ
                     EENSKKGNRTPFQYLSSSDSKISYETFKKHILNLAKGKGRISKTKKEYLLEERDINRFS
                     VQKDFINRNLVDTRYATRGLMNLLRSYFRVNNLDVKVKSINGGFTSFLRRKWKFKKERN
                     KGYKHHAEDALIIANADFIFKEWKKLDKAKKVMENQMFEEKQAESMPEIETEQEYKEIF
                     ITPHQIKHIKDFKDYKYSHRVDKKPNRELINDTLYSTRKDDKGNTLIVNNLNGLYDKDN
                     DKLKKLINKSPEKLLMYHHDPQTYQKLKLIMEQYGDEKNPLYKYYEETGNYLTKYSKKD
                     NGPVIKKIKYYGNKLNAHLDITDDYPNSRNKVVKLSLKPYRFDVYLDNGVYKFVTVKNL
                     DVIKKENYYEVNSKCYEEAKKLKKISNQAEFIASFYNNDLIKINGELYRVIGVNNDLLN
                     RIEVNMIDITYREYLENMNDKRPPRIIKTIASKTQSIKKYSTDILGNLYEVKSKKHPQI
                     IKKG"
     CDS             complement(7629..7649)
                     /codon_start=1
                     /product="nuclear localization signal of SV40 (simian virus
                     40) large T antigen"
                     /label=SV40 NLS
                     /translation="PKKKRKV"
     protein_bind    complement(7662..7695)
                     /label=loxP
                     /bound_moiety="Cre recombinase"
                     /note="Cre-mediated recombination occurs in the 8-bp core 
                     sequence (ATGTATGC) (Shaw et al., 2021)."
     protein_bind    complement(7696..7729)
                     /label=lox2272
                     /bound_moiety="Cre recombinase"
                     /note="Cre-mediated recombination occurs in the 8-bp core 
                     sequence (AAGTATCC) (Shaw et al., 2021). lox2272 sites are 
                     compatible with each other, but incompatible with loxP or 
                     loxN sites (Lee and Saito, 1988)."
     misc_feature    7740..8328
                     /label=WPRE
                     /note="woodchuck hepatitis virus posttranscriptional 
                     regulatory element"
     primer_bind     complement(7793..7813)
                     /label=WPRE-R
                     /note="WPRE, reverse primer"
     CDS             complement(8211..8222)
                     /codon_start=1
                     /product="Factor Xa recognition and cleavage site"
                     /label=Factor Xa site
                     /translation="IEGR"
     primer_bind     complement(8331..8347)
                     /label=KS primer
                     /note="common sequencing primer, one of multiple similar 
                     variants"
     primer_bind     complement(8332..8348)
                     /label=pBluescriptKS
                     /note="For pBluescript vector"
     primer_bind     complement(8349..8366)
                     /label=BGH-rev
                     /note="Bovine growth hormone terminator, reverse primer. 
                     Also called BGH reverse"
     polyA_signal    8355..8466
                     /label=bGH poly(A) signal
                     /note="bovine growth hormone polyadenylation signal"
     repeat_region   8570..8604
                     /label=piggyBac left (5') inverted repeat
                     /note="piggyBac transposon-specific inverted terminal 
                     repeat sequence (ITR)"
     primer_bind     complement(8627..8643)
                     /label=M13 rev
                     /note="common sequencing primer, one of multiple similar 
                     variants"
     primer_bind     complement(8627..8643)
                     /label=M13 Reverse
                     /note="In lacZ gene. Also called M13-rev"
     primer_bind     complement(8640..8662)
                     /label=M13/pUC Reverse
                     /note="In lacZ gene"
     protein_bind    8651..8667
                     /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(8675..8681,8682..8699,8700..8705))
                     /label=lac promoter
                     /note="promoter for the E. coli lac operon"
     protein_bind    8720..8741
                     /label=CAP binding site
                     /bound_moiety="E. coli catabolite activator protein"
                     /note="CAP binding activates transcription in the presence 
                     of cAMP."
     promoter        8800..8995
                     /label=SV40 promoter
                     /note="SV40 early promoter"
     rep_origin      8846..8981
                     /label=SV40 ori
                     /note="SV40 origin of replication"
     polyA_signal    9001..9135
                     /label=SV40 poly(A) signal
                     /note="SV40 polyadenylation signal"
     primer_bind     complement(9038..9057)
                     /label=SV40pA-R
                     /note="SV40 polyA, reverse primer"
     primer_bind     9092..9111
                     /label=EBV-rev
                     /note="SV40 polyA terminator, reverse primer"
     primer_bind     complement(9203..9220)
                     /label=L4440
                     /note="L4440 vector, forward primer"
     rep_origin      complement(9374..9962)
                     /direction=LEFT
                     /label=ori
                     /note="high-copy-number ColE1/pMB1/pBR322/pUC origin of 
                     replication"
     primer_bind     complement(9454..9473)
                     /label=pBR322ori-F
                     /note="pBR322 origin, forward primer"
     CDS             complement(join(10133..10924,10925..10993))
                     /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     10756..10775
                     /label=Amp-R
                     /note="Ampicillin resistance gene, reverse primer"
     promoter        complement(join(10994..11025,1..73))
                     /gene="bla"
                     /label=AmpR promoter
ORIGIN
        1 atcagggtta ttgtctcatg agcggataca tatttgaatg tatttagaaa aataaacaaa
       61 taggggttcc gcgcacattt ccccgaaaag tgccacctgg gtcgacttaa ccctagaaag
      121 ataatcatat tgtgacgtac gttaaagata atcatgcgta aaattgacgc atgtgtttta
      181 tcgctagtta ttaatagtaa tcaattacgg ggtcattagt tcatagccca tatatggagt
      241 tccgcgttac ataacttacg gtaaatggcc cgcctggctg accgcccaac gacccccgcc
      301 cattgacgtc aataatgacg tatgttccca tagtaacgcc aatagggact ttccattgac
      361 gtcaatgggt ggagtattta cggtaaactg cccacttggc agtacatcaa gtgtatcata
      421 tgccaagtac gccccctatt gacgtcaatg acggtaaatg gcccgcctgg cattatgccc
      481 agtacatgac cttatgggac tttcctactt ggcagtacat ctacgtatta gtcatcgcta
      541 ttaccatggt cgaggtgagc cccacgttct gcttcactct ccccatctcc cccccctccc
      601 cacccccaat tttgtattta tttatttttt aattattttg tgcagcgatg ggggcggggg
      661 gggggggggg ggggcggggc ggggcgaggg gcggggcggg gcgaggcgga gaggtgcggc
      721 ggcagccaat cagagcggcg cgctccgaaa gtttcctttt atggcgaggc ggcggcggcg
      781 gcggccctat aaaaagcgaa gcgcgcggcg ggcgggagtc gctgcgcgct gccttcgccc
      841 cgtgccccgc tccgccgccg cctcgcgccg cccgccccgg ctctgactga ccgcgttact
      901 cccacaggtg agcgggcggg acggcccttc tcctccgggc tgtaattagc gcttggttta
      961 atgacggctt gtttcttttc tgtggctgcg tgaaagcctt gaggggctcc gggagggccc
     1021 tttgtgcggg gggagcggct cggggggtgc gtgcgtgtgt gtgtgcgtgg ggagcgccgc
     1081 gtgcggctcc gcgctgcccg gcggctgtga gcgctgcggg cgcggcgcgg ggctttgtgc
     1141 gctccgcagt gtgcgcgagg ggagcgcggc cgggggcggt gccccgcggt gcgggggggg
     1201 ctgcgagggg aacaaaggct gcgtgcgggg tgtgtgcgtg ggggggtgag cagggggtgt
     1261 gggcgcgtcg gtcgggctgc aaccccccct gcacccccct ccccgagttg ctgagcacgg
     1321 cccggcttcg ggtgcggggc tccgtacggg gcgtggcgcg gggctcgccg tgccgggcgg
     1381 ggggtggcgg caggtggggg tgccgggcgg ggcggggccg cctcgggccg gggagggctc
     1441 gggggagggg cgcggcggcc cccggagcgc cggcggctgt cgaggcgcgg cgagccgcag
     1501 ccattgcctt ttatggtaat cgtgcgagag ggcgcaggga cttcctttgt cccaaatctg
     1561 tgcggagccg aaatctggga ggcgccgccg caccccctct agcgggcgcg gggcgaagcg
     1621 gtgcggcgcc ggcaggaagg aaatgggcgg ggagggcctt cgtgcgtcgc cgcgccgccg
     1681 tccccttctc cctctccagc ctcggggctg tccgcggggg gacggctgcc ttcggggggg
     1741 acggggcagg gcggggttcg gcttctggcg tgtgaccggc ggctctagag cctctgctaa
     1801 ccatgttcat gccttcttct ttttcctaca gctcctgggc aacgtgctgg ttattgtgct
     1861 gtctcatcat tttggcaaag ataacttcgt ataatgtatg ctatacgaag ttatgaagtt
     1921 cctattctct agaaagtata ggaacttcat ggccaccgag tacaagccca cggtgcgcct
     1981 cgccacccgc gacgacgtcc ccagggccgt acgcaccctc gccgccgcgt tcgccgacta
     2041 ccccgccacg cgccacaccg tcgatccgga ccgccacatc gagcgggtca ccgagctgca
     2101 agaactcttc ctcacgcgcg tcgggctcga catcggcaag gtgtgggtcg cggacgacgg
     2161 cgccgcggtg gcggtctgga ccacgccgga gagcgtcgaa gcgggggcgg tgttcgccga
     2221 gatcggcccg cgcatggccg agttgagcgg ttcccggctg gccgcgcagc aacagatgga
     2281 aggcctcctg gcgccgcacc ggcccaagga gcccgcgtgg ttcctggcca ccgtcggagt
     2341 ctcgcccgac caccagggca agggtctggg cagcgccgtc gtgctccccg gagtggaggc
     2401 ggccgagcgc gccggggtgc ccgccttcct ggagacctcc gcgccccgca acctcccctt
     2461 ctacgagcgg ctcggcttca ccgtcaccgc cgacgtcgag gtgcccgaag gaccgcgcac
     2521 ctggtgcatg acccgcaagc ccggtgccga attcggaagc ggagccacta acttctccct
     2581 gttgaaacaa gcaggggatg tcgaagagaa tcccgggcca cccaagaaga agaggaaggt
     2641 gggccccaag aagaagagga aggtgggccc caagaagaag aggaaggtgg gcctgcagcg
     2701 catcgccacc atggtgagca agggcgagga gctgttcacc ggggtggtgc ccatcctggt
     2761 cgagctggac ggcgacgtaa acggccacaa gttcagcgtg tccggcgagg gcgagggcga
     2821 tgccacctac ggcaagctga ccctgaagtt catctgcacc accggcaagc tgcccgtgcc
     2881 ctggcccacc ctcgtgacca ccctgaccta cggcgtgcag tgcttcagcc gctaccccga
     2941 ccacatgaag cagcacgact tcttcaagtc cgccatgccc gaaggctacg tccaggagcg
     3001 caccatcttc ttcaaggacg acggcaacta caagacccgc gccgaggtga agttcgaggg
     3061 cgacaccctg gtgaaccgca tcgagctgaa gggcatcgac ttcaaggagg acggcaacat
     3121 cctggggcac aagctggagt acaactacaa cagccacaac gtctatatca tggccgacaa
     3181 gcagaagaac ggcatcaagg tgaacttcaa gatccgccac aacatcgagg acggcagcgt
     3241 gcagctcgcc gaccactacc agcagaacac ccccatcggc gacggccccg tgctgctgcc
     3301 cgacaaccac tacctgagca cccagtccgc cctgagcaaa gaccccaacg agaagcgcga
     3361 tcacatggtc ctgctggagt tcgtgaccgc cgccgggatc actctcggca tggacgagct
     3421 gtacaagtaa agcgaagttc ctattctcta gaaagtatag gaacttcaat aaaagatctt
     3481 tattttcatt agatctgtgt gttggttttt tgtgtgataa cttcgtataa agtatcctat
     3541 acgaagttat ccggtgctta cttgtacagc tcgtccatgc cgccggtgga gtggcggccc
     3601 tcggcgcgtt cgtactgttc cacgatggtg tagtcctcgt tgtgggaggt gatgtccaac
     3661 ttgatgttga cgttgtaggc gccgggcagc tgcacgggct tcttggcctt gtaggtggtc
     3721 ttgacctcag cgtcgtagtg gccgccgtcc ttcagcttca gcctctgctt gatctcgccc
     3781 ttcagggcgc cgtcctcggg gtacatccgc tcggaggagg cctcccagcc catggtcttc
     3841 ttctgcatta cggggccgtc ggaggggaag ttggtgccgc gcagcttcac cttgtagatg
     3901 aactcgccgt cctgcaggga ggagtcctgg gtcacggtca ccacgccgcc gtcctcgaag
     3961 ttcatcacgc gctcccactt gaagccctcg gggaaggaca gcttcaagta gtcggggatg
     4021 tcggcggggt gcttcacgta ggccttggag ccgtacatga actgagggga caggatgtcc
     4081 caggcgaagg gcagggggcc acccttggtc accttcagct tggcggtctg ggtgccctcg
     4141 taggggcggc cctcgccctc gccctcgatc tcgaactcgt ggccgttcac ggagccctcc
     4201 atgtgcacct tgaagcgcat gaactccttg atgatggcca tgttatcctc ctcgcccttg
     4261 ctcacggtac ctggcccggg attctcttcg acatcccctg cttgtttcaa cagggagaag
     4321 ttagtggctc cgcttccgct agcgtaatct ggaacatcgt atgggtaagc gtaatctgga
     4381 acatcgtatg ggtaggatcc ctttttcttt tttgcctggc cggccttttt cgtggccgcc
     4441 ggccttttgc cctttttgat gatctgaggg tgcttcttag atttcacttc atacaggttg
     4501 cccagaatgt ctgtgctgta cttcttaatg ctctgggtct tggaggcgat tgtcttaatg
     4561 atcctggggg gcctcttgtc gttcatgttt tccaggtact cgcggtaggt gatgtcgatc
     4621 atgttcactt cgatccggtt cagcaggtcg ttgttcacgc cgatcactct atacagctcg
     4681 ccgttgatct tgatcagatc gttgttgtag aaggaggcga taaactcggc ctggttgctg
     4741 atcttcttca gcttcttagc ttcctcatag cacttgctat tcacttcgta gtagttttct
     4801 tttttgatca catccagatt cttcacggtc acgaacttgt acacgccatt gtccaggtac
     4861 acgtcgaatc tgtagggctt cagggacagc ttcacgacct tgtttctgct gttggggtag
     4921 tcgtcggtga tgtccagatg ggcgttcagt ttgttgccgt aatacttaat cttcttgatc
     4981 acggggccgt tgtccttttt ggagtacttg gtcaggtagt tcccggtttc ctcgtagtac
     5041 ttgtacaggg gattcttctc gtcgccgtac tgttccataa tcagcttcag tttctggtag
     5101 gtctgggggt cgtggtggta catcagcagc ttttcggggc tcttgttgat cagctttttc
     5161 agcttgtcat tgtccttgtc gtacaggccg ttcagattgt tcacgatcag ggtgttgccc
     5221 ttgtcgtcct tccgggtgga gtacagggtg tcgttaatca gctctctatt aggcttcttg
     5281 tccacccggt ggctgtactt gtagtccttg aagtccttaa tgtgcttgat ctggtggggg
     5341 gtgatgaaga tctctttgta ctcctgctcg gtttcgatct cgggcatgct ctcggcctgc
     5401 ttttcctcga acatctggtt ttccatcact tttttggcct tgtccagttt cttccactct
     5461 ttgaagatga aatcggcgtt ggcaatgatc agggcgtcct cggcgtggtg cttgtacccc
     5521 ttgttccgct ctttcttaaa cttccacttc cgccgcagaa agctggtgaa gccgccattg
     5581 atggacttca ctttcacgtc caggttgttc actctgaagt agctccgcag caggttcatc
     5641 aggcctctgg tggcgtatct ggtatccacc aggttccggt tgatgaagtc tttctgcacg
     5701 gagaacctgt tgatgtcccg ttcttccagc agatactctt tcttggtctt gctgattctg
     5761 cccttgccct tggccagatt caggatgtgc ttcttgaagg tttcgtagct gatcttgctg
     5821 tcgctgctgc tcaggtactg gaatggggtc cggttgccct tcttgctgtt ttcttcctgc
     5881 ttcacgagca ccttgttgtt gaagctgttg tcgaaggaca cgcttctggg gatgatgtgg
     5941 tccacctcat agttgaaggg gttgttcagc agatcttcca gagggatggc ttccaggctg
     6001 tacaggcact tgccttcctg catgtcgtgc agcttgatct tctcgatcag gtacttggcg
     6061 ttctctttgc cggtggtccg gatgatttcc tcgatccgct cgttggtctg ccggttccgc
     6121 ttctgcatct cgttgatcat tttctgggcg tccttggagt tcttctcgcg ggccagctcg
     6181 ataatgatgt cgttgggcag gccgtacttc ttgatgatgg cgttgatcac tttgatgctc
     6241 tggatgaagc ttctcttcac gacggggctc aggatgaagt cgtccaccag ggtggtgggg
     6301 atctctttct gctgggacag gtccaccttc ttgggcacca gcttcagccg gttgaagata
     6361 gcgatctggt tgtcgttggt gtgccacagc tcgtccagga tcaggttgat ggccttcagg
     6421 ctcaggttgt gggtgccggt atagcccttc agattagaga tctgctcgat ctcttcctgg
     6481 gtcagctcgg agttcagatt ggtcagttct tcctggatgt cctcgctgct ctggtagatg
     6541 gtcaggatct tggcaatctg atccagcagc tcggcgttct caataatctc tttccgggcg
     6601 gtaatgtcct tgatgtcgtg gtacaccttc aggttggtga actcgggctt gccggtgctg
     6661 gtcactctgt agcccttaat atcctcttcg ttcacgagga tttctttggc gatctgcttc
     6721 agggtgggct tcttcttctg cttgaacacg ttctcgatga tctggaactt ctcgtaatat
     6781 tccagcttct cgttctcgtc cctggtgatc acgagattgt tcaggtcgtt cagggcgttg
     6841 tacaggtcgg cgttgtaggc gtacttcacg ctccgcagtt cctcggggaa gtaggtgcag
     6901 tggcccatca gcatctcgta ccattctttg atgtccttcc agccgaaggg gctgccctcg
     6961 ccaggtccct catagtaggt ccgccgggtt tccagcaggt cgatgtaggt gtcgatgaag
     7021 ctctggtcca gctggtggta ggccttctgc accttcagca gctgtttggc ttctttcacg
     7081 tagtcgctgg tcttgaatct gttgatgctg ccccgcactt cgccgtcttt cttcagccgt
     7141 tccagctgca gttcggccac gtatttctct tccagggcct tgctgttccg gctgatctgc
     7201 tctttggtgg acagctcgtt gccggtgtcc tcttccacct cgttcacgtt gtgcacgcct
     7261 cttctcttgg ccaggtgcag cagggcggca gagaactctt cctcgctcag cttctggctc
     7321 aggcccttca ctctggcctc gtaggggttg atgccgctca gctcgctgtg gtcggtcagc
     7381 aggttgtagt cgaacagcag cttcttcact ctctggattc tatgccgcct ccgccgcttc
     7441 agccttctgg cgcctctctt gctccgcctg ccctcgttgt tttccacgtt ggcctctttg
     7501 aacagccgca cgccggcatc gatcacgtcc cgtgtctcgt agtcgatgat gccgtagccc
     7561 acgctggtga tgccgatgtc caggcccagg atgtagttcc gcttggctgc tgggactccg
     7621 tggataccga ccttccgctt cttctttggg gccatctcga gataacttcg tatagcatac
     7681 attatacgaa gttatataac ttcgtatagg atactttata cgaagttatg atatccgata
     7741 atcaacctct ggattacaaa atttgtgaaa gattgactgg tattcttaac tatgttgctc
     7801 cttttacgct atgtggatac gctgctttaa tgcctttgta tcatgctatt gcttcccgta
     7861 tggctttcat tttctcctcc ttgtataaat cctggttgct gtctctttat gaggagttgt
     7921 ggcccgttgt caggcaacgt ggcgtggtgt gcactgtgtt tgctgacgca acccccactg
     7981 gttggggcat tgccaccacc tgtcagctcc tttccgggac tttcgctttc cccctcccta
     8041 ttgccacggc ggaactcatc gccgcctgcc ttgcccgctg ctggacaggg gctcggctgt
     8101 tgggcactga caattccgtg gtgttgtcgg ggaaatcatc gtcctttcct tggctgctcg
     8161 cctgtgttgc cacctggatt ctgcgcggga cgtccttctg ctacgtccct tcggccctca
     8221 atccagcgga ccttccttcc cgcggcctgc tgccggctct gcggcctctt ccgcgtcttc
     8281 gccttcgccc tcagacgagt cggatctccc tttgggccgc ctccccgcat cgataccgtc
     8341 gacctcgacc tcgactgtgc cttctagttg ccagccatct gttgtttgcc cctcccccgt
     8401 gccttccttg accctggaag gtgccactcc cactgtcctt tcctaataaa atgaggaaat
     8461 tgcatcgcat tgtctgagta ggtgtcattc tattctgggg ggtggggtgg ggcaggacag
     8521 caagggggag gattgggaag acaataggag agagcaatat ttcaagatgc atgcgtcaat
     8581 tttacgcaga ctatctttct agggttaaaa gcttggcgta atcatggtca tagctgtttc
     8641 ctgtgtgaaa ttgttatccg ctcacaattc cacacaacat acgagccgga agcataaagt
     8701 gtaaagcctg gggtgcctaa tgagtgagct aactcacatt aattgcgttg cgctcactgc
     8761 ccgctttcca gtcgggaaac ctgtcgtgcc agcggatccg catctcaata agtcagcaac
     8821 catagtcccg cccctaactc cgcccatccc gcccctaact ccgcccagtt ccgcccattc
     8881 tccgccccat ggctgactaa ttttttttat ttatgcagag gccgaggccg cctcggcctc
     8941 tgagctattc cagaagtagt gaggaggctt ttttggaggc ctaggctttt gcaaaaagct
     9001 aacttgttta ttgcagctta taatggttac aaataaagca atagcatcac aaatttcaca
     9061 aataaagcat ttttttcact gcattctagt tgtggtttgt ccaaactcat caatgtatct
     9121 tatcatgtct ggatccgctg cattaatgaa tcggccaacg cgcggggaga ggcggtttgc
     9181 gtattgggcg ctcttccgct tcctcgctca ctgactcgct gcgctcggtc gttcggctgc
     9241 ggcgagcggt atcagctcac tcaaaggcgg taatacggtt atccacagaa tcaggggata
     9301 acgcaggaaa gaacatgtga gcaaaaggcc agcaaaaggc caggaaccgt aaaaaggccg
     9361 cgttgctggc gtttttccat aggctccgcc cccctgacga gcatcacaaa aatcgacgct
     9421 caagtcagag gtggcgaaac ccgacaggac tataaagata ccaggcgttt ccccctggaa
     9481 gctccctcgt gcgctctcct gttccgaccc tgccgcttac cggatacctg tccgcctttc
     9541 tcccttcggg aagcgtggcg ctttctcata gctcacgctg taggtatctc agttcggtgt
     9601 aggtcgttcg ctccaagctg ggctgtgtgc acgaaccccc cgttcagccc gaccgctgcg
     9661 ccttatccgg taactatcgt cttgagtcca acccggtaag acacgactta tcgccactgg
     9721 cagcagccac tggtaacagg attagcagag cgaggtatgt aggcggtgct acagagttct
     9781 tgaagtggtg gcctaactac ggctacacta gaagaacagt atttggtatc tgcgctctgc
     9841 tgaagccagt taccttcgga aaaagagttg gtagctcttg atccggcaaa caaaccaccg
     9901 ctggtagcgg tggttttttt gtttgcaagc agcagattac gcgcagaaaa aaaggatctc
     9961 aagaagatcc tttgatcttt tctacggggt ctgacgctca gtggaacgaa aactcacgtt
    10021 aagggatttt ggtcatgaga ttatcaaaaa ggatcttcac ctagatcctt ttaaattaaa
    10081 aatgaagttt taaatcaatc taaagtatat atgagtaaac ttggtctgac agttaccaat
    10141 gcttaatcag tgaggcacct atctcagcga tctgtctatt tcgttcatcc atagttgcct
    10201 gactccccgt cgtgtagata actacgatac gggagggctt accatctggc cccagtgctg
    10261 caatgatacc gcgagaccca cgctcaccgg ctccagattt atcagcaata aaccagccag
    10321 ccggaagggc cgagcgcaga agtggtcctg caactttatc cgcctccatc cagtctatta
    10381 attgttgccg ggaagctaga gtaagtagtt cgccagttaa tagtttgcgc aacgttgttg
    10441 ccattgctac aggcatcgtg gtgtcacgct cgtcgtttgg tatggcttca ttcagctccg
    10501 gttcccaacg atcaaggcga gttacatgat cccccatgtt gtgcaaaaaa gcggttagct
    10561 ccttcggtcc tccgatcgtt gtcagaagta agttggccgc agtgttatca ctcatggtta
    10621 tggcagcact gcataattct cttactgtca tgccatccgt aagatgcttt tctgtgactg
    10681 gtgagtactc aaccaagtca ttctgagaat agtgtatgcg gcgaccgagt tgctcttgcc
    10741 cggcgtcaat acgggataat accgcgccac atagcagaac tttaaaagtg ctcatcattg
    10801 gaaaacgttc ttcggggcga aaactctcaa ggatcttacc gctgttgaga tccagttcga
    10861 tgtaacccac tcgtgcaccc aactgatctt cagcatcttt tactttcacc agcgtttctg
    10921 ggtgagcaaa aacaggaagg caaaatgccg caaaaaaggg aataagggcg acacggaaat
    10981 gttgaatact catactcttc ctttttcaat attattgaag cattt
//
