LOCUS       sequence-330309-        8114 bp    DNA     circular SYN 14-APR-2025
DEFINITION  PiggyBac transposon vector constitutively driving PuroR-P2A-eGFP and
            Cre conditional expression of KASH nuclear membrane-bound mCherry. 
            Conditional insert flanked by MCS for cloning.
ACCESSION   .
VERSION     .
KEYWORDS    .
SOURCE      synthetic DNA construct
  ORGANISM  synthetic DNA construct
REFERENCE   1  (bases 1 to 8114)
  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 8114)
  AUTHORS   .
  TITLE     Direct Submission
  JOURNAL   Exported Apr 14, 2025 from SnapGene Server 8.0.1
            https://www.snapgene.com
COMMENT     SGRef: number: 1; type: "Journal Article"; journalName: "Sci Rep."; 
            date: "2022-03-14"; volume: "12(1):4369. doi"; pages: " 
            10.1038/s41598-022-07434-7"
COMMENT     Sequence Label: pMB953:PB-CAG-PuroR-nGFP-FLEx-mCherry-KASH-WPRE
FEATURES             Location/Qualifiers
     source          1..8114
                     /mol_type="other DNA"
                     /organism="synthetic DNA construct"
     intron          145..1161
                     /label=chimeric intron
                     /note="chimera between introns from chicken beta-actin and 
                     rabbit beta-globin"
     primer_bind     1085..1107
                     /label=pCAGGS-5
                     /note="Chimeric intron in CAG promoter, forward primer"
     primer_bind     1169..1188
                     /label=pCAG-F
                     /note="Rabbit beta-globin intron, for pCAG plasmids, 
                     forward primer"
     protein_bind    1211..1244
                     /label=loxP
                     /bound_moiety="Cre recombinase"
                     /note="Cre-mediated recombination occurs in the 8-bp core 
                     sequence (ATGTATGC) (Shaw et al., 2021)."
     protein_bind    1245..1278
                     /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             1279..1878
                     /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     1778..1798
                     /label=Puro-F
                     /note="Puromycin resistance gene, forward primer"
     CDS             1894..1950
                     /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             1951..1971
                     /codon_start=1
                     /product="nuclear localization signal of SV40 (simian virus
                     40) large T antigen"
                     /label=SV40 NLS
                     /translation="PKKKRKV"
     CDS             1975..1995
                     /codon_start=1
                     /product="nuclear localization signal of SV40 (simian virus
                     40) large T antigen"
                     /label=SV40 NLS
                     /translation="PKKKRKV"
     CDS             1999..2019
                     /codon_start=1
                     /product="nuclear localization signal of SV40 (simian virus
                     40) large T antigen"
                     /label=SV40 NLS
                     /translation="PKKKRKV"
     regulatory      2035..2044
                     /label=Kozak sequence
                     /note="vertebrate consensus sequence for strong initiation 
                     of translation (Kozak, 1987)"
                     /regulatory_class="other"
     CDS             join(2041..2043,2044..2046,2047..2760)
                     /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(2041..2043,2044..2046,2047..2757)
                     /codon_start=1
                     /product="enhanced GFP"
                     /label=EGFP
                     /translation="MVSKGEELFTGVVPILVELDGDVNGHKFSVSGEGEGDATYGKLTL
                     KFICTTGKLPVPWPTLVTTLTYGVQCFSRYPDHMKQHDFFKSAMPEGYVQERTIFFKDD
                     GNYKTRAEVKFEGDTLVNRIELKGIDFKEDGNILGHKLEYNYNSHNVYIMADKQKNGIK
                     VNFKIRHNIEDGSVQLADHYQQNTPIGDGPVLLPDNHYLSTQSALSKDPNEKRDHMVLL
                     EFVTAAGITLGMDELYK"
     primer_bind     complement(2086..2107)
                     /label=EGFP-N
                     /note="EGFP, reverse primer"
     primer_bind     complement(2347..2366)
                     /label=EXFP-R
                     /note="For distinguishing EGFP variants, reverse primer"
     primer_bind     2694..2715
                     /label=EGFP-C
                     /note="EGFP, forward primer"
     protein_bind    2764..2797
                     /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    2798..2846
                     /note="synthetic polyadenylation signal"
     protein_bind    2847..2880
                     /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(3165..3869)
                     /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(3440..3459)
                     /label=mCherry-F
                     /note="mCherry, forward primer"
     primer_bind     3709..3727
                     /label=mCherry-R
                     /note="mCherry, reverse primer"
     protein_bind    complement(3918..3951)
                     /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(3952..3985)
                     /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    3996..4584
                     /label=WPRE
                     /note="woodchuck hepatitis virus posttranscriptional 
                     regulatory element"
     primer_bind     complement(4049..4069)
                     /label=WPRE-R
                     /note="WPRE, reverse primer"
     CDS             complement(4467..4478)
                     /codon_start=1
                     /product="Factor Xa recognition and cleavage site"
                     /label=Factor Xa site
                     /translation="IEGR"
     primer_bind     complement(4587..4603)
                     /label=KS primer
                     /note="common sequencing primer, one of multiple similar 
                     variants"
     primer_bind     complement(4588..4604)
                     /label=pBluescriptKS
                     /note="For pBluescript vector"
     primer_bind     complement(4605..4622)
                     /label=BGH-rev
                     /note="Bovine growth hormone terminator, reverse primer. 
                     Also called BGH reverse"
     polyA_signal    4611..4722
                     /label=bGH poly(A) signal
                     /note="bovine growth hormone polyadenylation signal"
     repeat_region   4826..4860
                     /label=piggyBac left (5') inverted repeat
                     /note="piggyBac transposon-specific inverted terminal 
                     repeat sequence (ITR)"
     primer_bind     complement(4883..4899)
                     /label=M13 rev
                     /note="common sequencing primer, one of multiple similar 
                     variants"
     primer_bind     complement(4883..4899)
                     /label=M13 Reverse
                     /note="In lacZ gene. Also called M13-rev"
     primer_bind     complement(4896..4918)
                     /label=M13/pUC Reverse
                     /note="In lacZ gene"
     protein_bind    4907..4923
                     /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(4931..4937,4938..4955,4956..4961))
                     /label=lac promoter
                     /note="promoter for the E. coli lac operon"
     protein_bind    4976..4997
                     /label=CAP binding site
                     /bound_moiety="E. coli catabolite activator protein"
                     /note="CAP binding activates transcription in the presence 
                     of cAMP."
     promoter        5056..5251
                     /label=SV40 promoter
                     /note="SV40 early promoter"
     rep_origin      5102..5237
                     /label=SV40 ori
                     /note="SV40 origin of replication"
     polyA_signal    5257..5391
                     /label=SV40 poly(A) signal
                     /note="SV40 polyadenylation signal"
     primer_bind     complement(5294..5313)
                     /label=SV40pA-R
                     /note="SV40 polyA, reverse primer"
     primer_bind     5348..5367
                     /label=EBV-rev
                     /note="SV40 polyA terminator, reverse primer"
     primer_bind     complement(5459..5476)
                     /label=L4440
                     /note="L4440 vector, forward primer"
     rep_origin      complement(5630..6218)
                     /direction=LEFT
                     /label=ori
                     /note="high-copy-number ColE1/pMB1/pBR322/pUC origin of 
                     replication"
     primer_bind     complement(5710..5729)
                     /label=pBR322ori-F
                     /note="pBR322 origin, forward primer"
     CDS             complement(join(6389..7180,7181..7249))
                     /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     7012..7031
                     /label=Amp-R
                     /note="Ampicillin resistance gene, reverse primer"
     promoter        complement(7250..7354)
                     /gene="bla"
                     /label=AmpR promoter
     repeat_region   7392..7454
                     /label=piggyBac right (3') inverted repeat
                     /note="piggyBac transposon-specific inverted terminal 
                     repeat sequence (ITR)"
     enhancer        7526..7829
                     /label=CMV enhancer
                     /note="human cytomegalovirus immediate early enhancer"
ORIGIN
        1 ggggcggggc ggggcgaggg gcggggcggg gcgaggcgga gaggtgcggc ggcagccaat
       61 cagagcggcg cgctccgaaa gtttcctttt atggcgaggc ggcggcggcg gcggccctat
      121 aaaaagcgaa gcgcgcggcg ggcgggagtc gctgcgcgct gccttcgccc cgtgccccgc
      181 tccgccgccg cctcgcgccg cccgccccgg ctctgactga ccgcgttact cccacaggtg
      241 agcgggcggg acggcccttc tcctccgggc tgtaattagc gcttggttta atgacggctt
      301 gtttcttttc tgtggctgcg tgaaagcctt gaggggctcc gggagggccc tttgtgcggg
      361 gggagcggct cggggggtgc gtgcgtgtgt gtgtgcgtgg ggagcgccgc gtgcggctcc
      421 gcgctgcccg gcggctgtga gcgctgcggg cgcggcgcgg ggctttgtgc gctccgcagt
      481 gtgcgcgagg ggagcgcggc cgggggcggt gccccgcggt gcgggggggg ctgcgagggg
      541 aacaaaggct gcgtgcgggg tgtgtgcgtg ggggggtgag cagggggtgt gggcgcgtcg
      601 gtcgggctgc aaccccccct gcacccccct ccccgagttg ctgagcacgg cccggcttcg
      661 ggtgcggggc tccgtacggg gcgtggcgcg gggctcgccg tgccgggcgg ggggtggcgg
      721 caggtggggg tgccgggcgg ggcggggccg cctcgggccg gggagggctc gggggagggg
      781 cgcggcggcc cccggagcgc cggcggctgt cgaggcgcgg cgagccgcag ccattgcctt
      841 ttatggtaat cgtgcgagag ggcgcaggga cttcctttgt cccaaatctg tgcggagccg
      901 aaatctggga ggcgccgccg caccccctct agcgggcgcg gggcgaagcg gtgcggcgcc
      961 ggcaggaagg aaatgggcgg ggagggcctt cgtgcgtcgc cgcgccgccg tccccttctc
     1021 cctctccagc ctcggggctg tccgcggggg gacggctgcc ttcggggggg acggggcagg
     1081 gcggggttcg gcttctggcg tgtgaccggc ggctctagag cctctgctaa ccatgttcat
     1141 gccttcttct ttttcctaca gctcctgggc aacgtgctgg ttattgtgct gtctcatcat
     1201 tttggcaaag ataacttcgt ataatgtatg ctatacgaag ttatgaagtt cctattctct
     1261 agaaagtata ggaacttcat ggccaccgag tacaagccca cggtgcgcct cgccacccgc
     1321 gacgacgtcc ccagggccgt acgcaccctc gccgccgcgt tcgccgacta ccccgccacg
     1381 cgccacaccg tcgatccgga ccgccacatc gagcgggtca ccgagctgca agaactcttc
     1441 ctcacgcgcg tcgggctcga catcggcaag gtgtgggtcg cggacgacgg cgccgcggtg
     1501 gcggtctgga ccacgccgga gagcgtcgaa gcgggggcgg tgttcgccga gatcggcccg
     1561 cgcatggccg agttgagcgg ttcccggctg gccgcgcagc aacagatgga aggcctcctg
     1621 gcgccgcacc ggcccaagga gcccgcgtgg ttcctggcca ccgtcggagt ctcgcccgac
     1681 caccagggca agggtctggg cagcgccgtc gtgctccccg gagtggaggc ggccgagcgc
     1741 gccggggtgc ccgccttcct ggagacctcc gcgccccgca acctcccctt ctacgagcgg
     1801 ctcggcttca ccgtcaccgc cgacgtcgag gtgcccgaag gaccgcgcac ctggtgcatg
     1861 acccgcaagc ccggtgccga attcggaagc ggagccacta acttctccct gttgaaacaa
     1921 gcaggggatg tcgaagagaa tcccgggcca cccaagaaga agaggaaggt gggccccaag
     1981 aagaagagga aggtgggccc caagaagaag aggaaggtgg gcctgcagcg catcgccacc
     2041 atggtgagca agggcgagga gctgttcacc ggggtggtgc ccatcctggt cgagctggac
     2101 ggcgacgtaa acggccacaa gttcagcgtg tccggcgagg gcgagggcga tgccacctac
     2161 ggcaagctga ccctgaagtt catctgcacc accggcaagc tgcccgtgcc ctggcccacc
     2221 ctcgtgacca ccctgaccta cggcgtgcag tgcttcagcc gctaccccga ccacatgaag
     2281 cagcacgact tcttcaagtc cgccatgccc gaaggctacg tccaggagcg caccatcttc
     2341 ttcaaggacg acggcaacta caagacccgc gccgaggtga agttcgaggg cgacaccctg
     2401 gtgaaccgca tcgagctgaa gggcatcgac ttcaaggagg acggcaacat cctggggcac
     2461 aagctggagt acaactacaa cagccacaac gtctatatca tggccgacaa gcagaagaac
     2521 ggcatcaagg tgaacttcaa gatccgccac aacatcgagg acggcagcgt gcagctcgcc
     2581 gaccactacc agcagaacac ccccatcggc gacggccccg tgctgctgcc cgacaaccac
     2641 tacctgagca cccagtccgc cctgagcaaa gaccccaacg agaagcgcga tcacatggtc
     2701 ctgctggagt tcgtgaccgc cgccgggatc actctcggca tggacgagct gtacaagtaa
     2761 agcgaagttc ctattctcta gaaagtatag gaacttcaat aaaagatctt tattttcatt
     2821 agatctgtgt gttggttttt tgtgtgataa cttcgtataa agtatcctat acgaagttat
     2881 ccggtgcggc cgctagccta ggtgggaggt ggcccgttgg tgtaccgcag catggggtag
     2941 aaggatcggg caaagttgtt ggcctgggtg cagctgtagt catcttcaga ggcaggtagc
     3001 aggcaggcca ggagcagcag cagcagcaga agcagctgca acggtagcgc tgccctgatc
     3061 acccttgaga ggaaggagcg tctcggctgg gagctgccgg ggctgtcgag gtggggcatc
     3121 ctgctgtctg tctcctcctc ctcctctcga gatctgagtc cggacttgta cagctcgtcc
     3181 atgccgccgg tggagtggcg gccctcggcg cgttcgtact gttccacgat ggtgtagtcc
     3241 tcgttgtggg aggtgatgtc caacttgatg ttgacgttgt aggcgccggg cagctgcacg
     3301 ggcttcttgg ccttgtaggt ggtcttgacc tcagcgtcgt agtggccgcc gtccttcagc
     3361 ttcagcctct gcttgatctc gcccttcagg gcgccgtcct cggggtacat ccgctcggag
     3421 gaggcctccc agcccatggt cttcttctgc attacggggc cgtcggaggg gaagttggtg
     3481 ccgcgcagct tcaccttgta gatgaactcg ccgtcctgca gggaggagtc ctgggtcacg
     3541 gtcaccacgc cgccgtcctc gaagttcatc acgcgctccc acttgaagcc ctcggggaag
     3601 gacagcttca agtagtcggg gatgtcggcg gggtgcttca cgtaggcctt ggagccgtac
     3661 atgaactgag gggacaggat gtcccaggcg aagggcaggg ggccaccctt ggtcaccttc
     3721 agcttggcgg tctgggtgcc ctcgtagggg cggccctcgc cctcgccctc gatctcgaac
     3781 tcgtggccgt tcacggagcc ctccatgtgc accttgaagc gcatgaactc cttgatgatg
     3841 gccatgttat cctcctcgcc cttgctcacg gtggcgtcga ctctagagga tccggtacca
     3901 ccggtacgcg tctcgagata acttcgtata gcatacatta tacgaagtta tataacttcg
     3961 tataggatac tttatacgaa gttatgatat ccgataatca acctctggat tacaaaattt
     4021 gtgaaagatt gactggtatt cttaactatg ttgctccttt tacgctatgt ggatacgctg
     4081 ctttaatgcc tttgtatcat gctattgctt cccgtatggc tttcattttc tcctccttgt
     4141 ataaatcctg gttgctgtct ctttatgagg agttgtggcc cgttgtcagg caacgtggcg
     4201 tggtgtgcac tgtgtttgct gacgcaaccc ccactggttg gggcattgcc accacctgtc
     4261 agctcctttc cgggactttc gctttccccc tccctattgc cacggcggaa ctcatcgccg
     4321 cctgccttgc ccgctgctgg acaggggctc ggctgttggg cactgacaat tccgtggtgt
     4381 tgtcggggaa atcatcgtcc tttccttggc tgctcgcctg tgttgccacc tggattctgc
     4441 gcgggacgtc cttctgctac gtcccttcgg ccctcaatcc agcggacctt ccttcccgcg
     4501 gcctgctgcc ggctctgcgg cctcttccgc gtcttcgcct tcgccctcag acgagtcgga
     4561 tctccctttg ggccgcctcc ccgcatcgat accgtcgacc tcgacctcga ctgtgccttc
     4621 tagttgccag ccatctgttg tttgcccctc ccccgtgcct tccttgaccc tggaaggtgc
     4681 cactcccact gtcctttcct aataaaatga ggaaattgca tcgcattgtc tgagtaggtg
     4741 tcattctatt ctggggggtg gggtggggca ggacagcaag ggggaggatt gggaagacaa
     4801 taggagagag caatatttca agatgcatgc gtcaatttta cgcagactat ctttctaggg
     4861 ttaaaagctt ggcgtaatca tggtcatagc tgtttcctgt gtgaaattgt tatccgctca
     4921 caattccaca caacatacga gccggaagca taaagtgtaa agcctggggt gcctaatgag
     4981 tgagctaact cacattaatt gcgttgcgct cactgcccgc tttccagtcg ggaaacctgt
     5041 cgtgccagcg gatccgcatc tcaataagtc agcaaccata gtcccgcccc taactccgcc
     5101 catcccgccc ctaactccgc ccagttccgc ccattctccg ccccatggct gactaatttt
     5161 ttttatttat gcagaggccg aggccgcctc ggcctctgag ctattccaga agtagtgagg
     5221 aggctttttt ggaggcctag gcttttgcaa aaagctaact tgtttattgc agcttataat
     5281 ggttacaaat aaagcaatag catcacaaat ttcacaaata aagcattttt ttcactgcat
     5341 tctagttgtg gtttgtccaa actcatcaat gtatcttatc atgtctggat ccgctgcatt
     5401 aatgaatcgg ccaacgcgcg gggagaggcg gtttgcgtat tgggcgctct tccgcttcct
     5461 cgctcactga ctcgctgcgc tcggtcgttc ggctgcggcg agcggtatca gctcactcaa
     5521 aggcggtaat acggttatcc acagaatcag gggataacgc aggaaagaac atgtgagcaa
     5581 aaggccagca aaaggccagg aaccgtaaaa aggccgcgtt gctggcgttt ttccataggc
     5641 tccgcccccc tgacgagcat cacaaaaatc gacgctcaag tcagaggtgg cgaaacccga
     5701 caggactata aagataccag gcgtttcccc ctggaagctc cctcgtgcgc tctcctgttc
     5761 cgaccctgcc gcttaccgga tacctgtccg cctttctccc ttcgggaagc gtggcgcttt
     5821 ctcatagctc acgctgtagg tatctcagtt cggtgtaggt cgttcgctcc aagctgggct
     5881 gtgtgcacga accccccgtt cagcccgacc gctgcgcctt atccggtaac tatcgtcttg
     5941 agtccaaccc ggtaagacac gacttatcgc cactggcagc agccactggt aacaggatta
     6001 gcagagcgag gtatgtaggc ggtgctacag agttcttgaa gtggtggcct aactacggct
     6061 acactagaag aacagtattt ggtatctgcg ctctgctgaa gccagttacc ttcggaaaaa
     6121 gagttggtag ctcttgatcc ggcaaacaaa ccaccgctgg tagcggtggt ttttttgttt
     6181 gcaagcagca gattacgcgc agaaaaaaag gatctcaaga agatcctttg atcttttcta
     6241 cggggtctga cgctcagtgg aacgaaaact cacgttaagg gattttggtc atgagattat
     6301 caaaaaggat cttcacctag atccttttaa attaaaaatg aagttttaaa tcaatctaaa
     6361 gtatatatga gtaaacttgg tctgacagtt accaatgctt aatcagtgag gcacctatct
     6421 cagcgatctg tctatttcgt tcatccatag ttgcctgact ccccgtcgtg tagataacta
     6481 cgatacggga gggcttacca tctggcccca gtgctgcaat gataccgcga gacccacgct
     6541 caccggctcc agatttatca gcaataaacc agccagccgg aagggccgag cgcagaagtg
     6601 gtcctgcaac tttatccgcc tccatccagt ctattaattg ttgccgggaa gctagagtaa
     6661 gtagttcgcc agttaatagt ttgcgcaacg ttgttgccat tgctacaggc atcgtggtgt
     6721 cacgctcgtc gtttggtatg gcttcattca gctccggttc ccaacgatca aggcgagtta
     6781 catgatcccc catgttgtgc aaaaaagcgg ttagctcctt cggtcctccg atcgttgtca
     6841 gaagtaagtt ggccgcagtg ttatcactca tggttatggc agcactgcat aattctctta
     6901 ctgtcatgcc atccgtaaga tgcttttctg tgactggtga gtactcaacc aagtcattct
     6961 gagaatagtg tatgcggcga ccgagttgct cttgcccggc gtcaatacgg gataataccg
     7021 cgccacatag cagaacttta aaagtgctca tcattggaaa acgttcttcg gggcgaaaac
     7081 tctcaaggat cttaccgctg ttgagatcca gttcgatgta acccactcgt gcacccaact
     7141 gatcttcagc atcttttact ttcaccagcg tttctgggtg agcaaaaaca ggaaggcaaa
     7201 atgccgcaaa aaagggaata agggcgacac ggaaatgttg aatactcata ctcttccttt
     7261 ttcaatatta ttgaagcatt tatcagggtt attgtctcat gagcggatac atatttgaat
     7321 gtatttagaa aaataaacaa ataggggttc cgcgcacatt tccccgaaaa gtgccacctg
     7381 ggtcgactta accctagaaa gataatcata ttgtgacgta cgttaaagat aatcatgcgt
     7441 aaaattgacg catgtgtttt atcgctagtt attaatagta atcaattacg gggtcattag
     7501 ttcatagccc atatatggag ttccgcgtta cataacttac ggtaaatggc ccgcctggct
     7561 gaccgcccaa cgacccccgc ccattgacgt caataatgac gtatgttccc atagtaacgc
     7621 caatagggac tttccattga cgtcaatggg tggagtattt acggtaaact gcccacttgg
     7681 cagtacatca agtgtatcat atgccaagta cgccccctat tgacgtcaat gacggtaaat
     7741 ggcccgcctg gcattatgcc cagtacatga ccttatggga ctttcctact tggcagtaca
     7801 tctacgtatt agtcatcgct attaccatgg tcgaggtgag ccccacgttc tgcttcactc
     7861 tccccatctc ccccccctcc ccacccccaa ttttgtattt atttattttt taattatttt
     7921 gtgcagcgat gggggcgggg gggggggggg gggggcgcgc cggggggggg gggggggggg
     7981 gggggggggg gggggggggg gggggggggg gggggggggg gggggggggg gggggggggg
     8041 gggggggggg gggggggggg gggggggggg gggggggggg gggggggggg gggggggggg
     8101 gggggggggg gggg
//
