LOCUS       sequence-373706-       11227 bp    DNA     circular SYN 15-DEC-2025
DEFINITION  FKBP12 (F36V mutant) degron-tagged KRAB-dCas9 fused with tagBFP, 
            expressed under EF1-alpha promoter in a piggyBac plasmid. Expresses 
            Blasticidin resistance under PGK promoter..
ACCESSION   .
VERSION     .
KEYWORDS    .
SOURCE      synthetic DNA construct
  ORGANISM  synthetic DNA construct
REFERENCE   1  (bases 1 to 11227)
  AUTHORS   Noviello G, Gjaltema RAF, Schulz EG
  TITLE     CasTuner is a degron and CRISPR/Cas-based toolkit for analog tuning 
            of endogenous gene expression.
  JOURNAL   Nat Commun. 2023 Jun 3;14(1):3225. doi: 10.1038/s41467-023-38909-4.
  PUBMED    37270532
REFERENCE   2  (bases 1 to 11227)
  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: "Nat 
            Commun."; date: "2023-06-3"; volume: "14(1):3225. doi"; pages: " 
            10.1038/s41467-023-38909-4"
COMMENT     Sequence Label: 
            pSLPB2B-FKBP12_F36V-KRAB-SpdCas9-tagBFP-PGK-Blasticidin
FEATURES             Location/Qualifiers
     source          1..11227
                     /mol_type="other DNA"
                     /organism="synthetic DNA construct"
     CDS             join(46..69,70..72,73..2559,2560..2562,2563..4146)
                     /codon_start=1
                     /product="catalytically dead mutant of the Cas9 
                     endonuclease from the Streptococcus pyogenes Type II 
                     CRISPR/Cas system"
                     /label=dCas9
                     /note="RNA-guided DNA-binding protein that lacks 
                     endonuclease activity due to the D10A mutation in the RuvC 
                     catalytic domain and the H840A mutation in the HNH 
                     catalytic domain"
                     /translation="DKKYSIGLAIGTNSVGWAVITDEYKVPSKKFKVLGNTDRHSIKKN
                     LIGALLFDSGETAEATRLKRTARRRYTRRKNRICYLQEIFSNEMAKVDDSFFHRLEESF
                     LVEEDKKHERHPIFGNIVDEVAYHEKYPTIYHLRKKLVDSTDKADLRLIYLALAHMIKF
                     RGHFLIEGDLNPDNSDVDKLFIQLVQTYNQLFEENPINASGVDAKAILSARLSKSRRLE
                     NLIAQLPGEKKNGLFGNLIALSLGLTPNFKSNFDLAEDAKLQLSKDTYDDDLDNLLAQI
                     GDQYADLFLAAKNLSDAILLSDILRVNTEITKAPLSASMIKRYDEHHQDLTLLKALVRQ
                     QLPEKYKEIFFDQSKNGYAGYIDGGASQEEFYKFIKPILEKMDGTEELLVKLNREDLLR
                     KQRTFDNGSIPHQIHLGELHAILRRQEDFYPFLKDNREKIEKILTFRIPYYVGPLARGN
                     SRFAWMTRKSEETITPWNFEEVVDKGASAQSFIERMTNFDKNLPNEKVLPKHSLLYEYF
                     TVYNELTKVKYVTEGMRKPAFLSGEQKKAIVDLLFKTNRKVTVKQLKEDYFKKIECFDS
                     VEISGVEDRFNASLGTYHDLLKIIKDKDFLDNEENEDILEDIVLTLTLFEDREMIEERL
                     KTYAHLFDDKVMKQLKRRRYTGWGRLSRKLINGIRDKQSGKTILDFLKSDGFANRNFMQ
                     LIHDDSLTFKEDIQKAQVSGQGDSLHEHIANLAGSPAIKKGILQTVKVVDELVKVMGRH
                     KPENIVIEMARENQTTQKGQKNSRERMKRIEEGIKELGSQILKEHPVENTQLQNEKLYL
                     YYLQNGRDMYVDQELDINRLSDYDVDAIVPQSFLKDDSIDNKVLTRSDKNRGKSDNVPS
                     EEVVKKMKNYWRQLLNAKLITQRKFDNLTKAERGGLSELDKAGFIKRQLVETRQITKHV
                     AQILDSRMNTKYDENDKLIREVKVITLKSKLVSDFRKDFQFYKVREINNYHHAHDAYLN
                     AVVGTALIKKYPKLESEFVYGDYKVYDVRKMIAKSEQEIGKATAKYFFYSNIMNFFKTE
                     ITLANGEIRKRPLIETNGETGEIVWDKGRDFATVRKVLSMPQVNIVKKTEVQTGGFSKE
                     SILPKRNSDKLIARKKDWDPKKYGGFDSPTVAYSVLVVAKVEKGKSKKLKSVKELLGIT
                     IMERSSFEKNPIDFLEAKGYKEVKKDLIIKLPKYSLFELENGRKRMLASAGELQKGNEL
                     ALPSKYVNFLYLASHYEKLKGSPEDNEQKQLFVEQHKHYLDEIIEQISEFSKRVILADA
                     NLDKVLSAYNKHRDKPIREQAENIIHLFTLTNLGAPAAFKYFDTTIDRKRYTSTKEVLD
                     ATLIHQSITGLYETRIDLSQLGGD"
     CDS             46..4146
                     /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="DKKYSIGLAIGTNSVGWAVITDEYKVPSKKFKVLGNTDRHSIKKN
                     LIGALLFDSGETAEATRLKRTARRRYTRRKNRICYLQEIFSNEMAKVDDSFFHRLEESF
                     LVEEDKKHERHPIFGNIVDEVAYHEKYPTIYHLRKKLVDSTDKADLRLIYLALAHMIKF
                     RGHFLIEGDLNPDNSDVDKLFIQLVQTYNQLFEENPINASGVDAKAILSARLSKSRRLE
                     NLIAQLPGEKKNGLFGNLIALSLGLTPNFKSNFDLAEDAKLQLSKDTYDDDLDNLLAQI
                     GDQYADLFLAAKNLSDAILLSDILRVNTEITKAPLSASMIKRYDEHHQDLTLLKALVRQ
                     QLPEKYKEIFFDQSKNGYAGYIDGGASQEEFYKFIKPILEKMDGTEELLVKLNREDLLR
                     KQRTFDNGSIPHQIHLGELHAILRRQEDFYPFLKDNREKIEKILTFRIPYYVGPLARGN
                     SRFAWMTRKSEETITPWNFEEVVDKGASAQSFIERMTNFDKNLPNEKVLPKHSLLYEYF
                     TVYNELTKVKYVTEGMRKPAFLSGEQKKAIVDLLFKTNRKVTVKQLKEDYFKKIECFDS
                     VEISGVEDRFNASLGTYHDLLKIIKDKDFLDNEENEDILEDIVLTLTLFEDREMIEERL
                     KTYAHLFDDKVMKQLKRRRYTGWGRLSRKLINGIRDKQSGKTILDFLKSDGFANRNFMQ
                     LIHDDSLTFKEDIQKAQVSGQGDSLHEHIANLAGSPAIKKGILQTVKVVDELVKVMGRH
                     KPENIVIEMARENQTTQKGQKNSRERMKRIEEGIKELGSQILKEHPVENTQLQNEKLYL
                     YYLQNGRDMYVDQELDINRLSDYDVDAIVPQSFLKDDSIDNKVLTRSDKNRGKSDNVPS
                     EEVVKKMKNYWRQLLNAKLITQRKFDNLTKAERGGLSELDKAGFIKRQLVETRQITKHV
                     AQILDSRMNTKYDENDKLIREVKVITLKSKLVSDFRKDFQFYKVREINNYHHAHDAYLN
                     AVVGTALIKKYPKLESEFVYGDYKVYDVRKMIAKSEQEIGKATAKYFFYSNIMNFFKTE
                     ITLANGEIRKRPLIETNGETGEIVWDKGRDFATVRKVLSMPQVNIVKKTEVQTGGFSKE
                     SILPKRNSDKLIARKKDWDPKKYGGFDSPTVAYSVLVVAKVEKGKSKKLKSVKELLGIT
                     IMERSSFEKNPIDFLEAKGYKEVKKDLIIKLPKYSLFELENGRKRMLASAGELQKGNEL
                     ALPSKYVNFLYLASHYEKLKGSPEDNEQKQLFVEQHKHYLDEIIEQISEFSKRVILADA
                     NLDKVLSAYNKHRDKPIREQAENIIHLFTLTNLGAPAAFKYFDTTIDRKRYTSTKEVLD
                     ATLIHQSITGLYETRIDLSQLGGD"
     CDS             4147..4167
                     /codon_start=1
                     /product="nuclear localization signal of SV40 (simian virus
                     40) large T antigen"
                     /label=SV40 NLS
                     /translation="PKKKRKV"
     CDS             4168..4185
                     /codon_start=1
                     /product="6xHis affinity tag"
                     /label=6xHis
                     /translation="HHHHHH"
     CDS             4240..4941
                     /codon_start=1
                     /product="monomeric blue fluorescent protein"
                     /label=TagBFP
                     /note="mammalian codon-optimized"
                     /translation="MSELIKENMHMKLYMEGTVDNHHFKCTSEGEGKPYEGTQTMRIKV
                     VEGGPLPFAFDILATSFLYGSKTFINHTQGIPDFFKQSFPEGFTWERVTTYEDGGVLTA
                     TQDTSLQDGCLIYNVKIRGVNFTSNGPVMQKKTLGWEAFTETLYPADGGLEGRNDMALK
                     LVGGSHLIANIKTTYRSKKPAKNLKMPGVYYVDYRLERIKEANNETYVEQHEVAVARYC
                     DLPSKLGHKLN"
     polyA_signal    4956..5180
                     /label=bGH poly(A) signal
                     /note="bovine growth hormone polyadenylation signal"
     primer_bind     5531..5550
                     /label=mPGK-F
                     /note="Mouse PGK promoter, forward primer"
     CDS             5606..6004
                     /codon_start=1
                     /gene="Aspergillus terreus BSD"
                     /product="blasticidin S deaminase"
                     /label=BSD
                     /note="confers resistance to blasticidin"
                     /translation="MAKPLSQEESTLIERATATINSIPISEDYSVASAALSSDGRIFTG
                     VNVYHFTGGPCAELVVLGTAAAAAAGNLTCIVAIGNENRGILSPCGRCRQVLLDLHPGI
                     KAIVKDSDGQPTAVGIRELLPSGYVWEG"
     misc_feature    6005..6593
                     /label=WPRE
                     /note="woodchuck hepatitis virus posttranscriptional 
                     regulatory element"
     primer_bind     complement(6058..6078)
                     /label=WPRE-R
                     /note="WPRE, reverse primer"
     CDS             complement(6476..6487)
                     /codon_start=1
                     /product="Factor Xa recognition and cleavage site"
                     /label=Factor Xa site
                     /translation="IEGR"
     repeat_region   6776..6838
                     /label=piggyBac right (3') inverted repeat
                     /note="piggyBac transposon-specific inverted terminal 
                     repeat sequence (ITR)"
     primer_bind     complement(6868..6888)
                     /label=T3
                     /note="T3 promoter, forward primer"
     promoter        complement(6868..6886)
                     /label=T3 promoter
                     /note="promoter for bacteriophage T3 RNA polymerase"
     primer_bind     complement(6907..6923)
                     /label=M13 rev
                     /note="common sequencing primer, one of multiple similar 
                     variants"
     primer_bind     complement(6907..6923)
                     /label=M13 Reverse
                     /note="In lacZ gene. Also called M13-rev"
     primer_bind     complement(6920..6942)
                     /label=M13/pUC Reverse
                     /note="In lacZ gene"
     protein_bind    6931..6947
                     /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(6955..6961,6962..6979,6980..6985))
                     /label=lac promoter
                     /note="promoter for the E. coli lac operon"
     protein_bind    7000..7021
                     /label=CAP binding site
                     /bound_moiety="E. coli catabolite activator protein"
                     /note="CAP binding activates transcription in the presence 
                     of cAMP."
     primer_bind     complement(7138..7155)
                     /label=L4440
                     /note="L4440 vector, forward primer"
     rep_origin      complement(7309..7897)
                     /direction=LEFT
                     /label=ori
                     /note="high-copy-number ColE1/pMB1/pBR322/pUC origin of 
                     replication"
     primer_bind     complement(7389..7408)
                     /label=pBR322ori-F
                     /note="pBR322 origin, forward primer"
     CDS             complement(join(8068..8859,8860..8928))
                     /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     8691..8710
                     /label=Amp-R
                     /note="Ampicillin resistance gene, reverse primer"
     promoter        complement(8929..9033)
                     /gene="bla"
                     /label=AmpR promoter
     rep_origin      complement(9059..9514)
                     /direction=LEFT
                     /label=f1 ori
                     /note="f1 bacteriophage origin of replication; arrow 
                     indicates direction of (+) strand synthesis"
     primer_bind     complement(9196..9217)
                     /label=F1ori-F
                     /note="F1 origin, forward primer"
     primer_bind     9408..9427
                     /label=F1ori-R
                     /note="F1 origin, reverse primer"
     primer_bind     9641..9663
                     /label=M13/pUC Forward
                     /note="In lacZ gene"
     primer_bind     9655..9672
                     /label=M13 Forward
                     /note="In lacZ gene. Also called M13-F20 or M13 (-21) 
                     Forward"
     primer_bind     9656..9672
                     /label=M13 fwd
                     /note="common sequencing primer, one of multiple similar 
                     variants"
     primer_bind     9682..9701
                     /label=T7
                     /note="T7 promoter, forward primer"
     promoter        9682..9700
                     /label=T7 promoter
                     /note="promoter for bacteriophage T7 RNA polymerase"
     repeat_region   9716..9750
                     /label=piggyBac left (5') inverted repeat
                     /note="piggyBac transposon-specific inverted terminal 
                     repeat sequence (ITR)"
     promoter        10072..10283
                     /label=EF-1-alpha core promoter
                     /note="core promoter for human elongation factor 
                     EF-1-alpha"
     LTR             10296..10564
                     /label=5' LTR (truncated)
                     /note="truncated 5' long terminal repeat (LTR) from human 
                     T-cell leukemia virus (HTLV) type 1"
     CDS             10616..10636
                     /codon_start=1
                     /product="nuclear localization signal of SV40 (simian virus
                     40) large T antigen"
                     /label=SV40 NLS
                     /translation="PKKKRKV"
     CDS             10646..10966
                     /codon_start=1
                     /product="F36V mutant of FK506-binding protein FKBP12"
                     /label=FKBP-V
                     /note="binds synthetic ligands that do not bind wild-type 
                     FKBP"
                     /translation="GVQVETISPGDGRTFPKRGQTCVVHYTGMLEDGKKVDSSRDRNKP
                     FKFMLGKQEVIRGWEEGVAQMSVGQRAKLTISPDYAYGATGHPGIIPPHATLVFDVELL
                     KLE"
     CDS             11042..11227
                     /codon_start=1
                     /product="Kruppel-associated box (KRAB) transcriptional 
                     repression domain from the human zinc finger protein ZNF10 
                     (Margolin et al., 1994)"
                     /label=KRAB
                     /translation="RTLVTFKDVFVDFTREEWKLLDTAQQIVYRNVMLENYKNLVSLGY
                     QLTKPDVILRLEKGEEP"
ORIGIN
        1 ggcggatctg gaggtagtgg tggatctgga ggttctggtg gctccgacaa gaagtacagc
       61 atcggcctgg ccatcggcac caactctgtg ggctgggccg tgatcaccga cgagtacaag
      121 gtgcccagca agaaattcaa ggtgctgggc aacaccgacc ggcacagcat caagaagaac
      181 ctgatcggcg ccctgctgtt cgacagcgga gaaacagccg aggccacccg gctgaagaga
      241 accgccagaa gaagatacac cagacggaag aaccggatct gctatctgca agagatcttc
      301 agcaacgaga tggccaaggt ggacgacagc ttcttccaca gactggaaga gtccttcctg
      361 gtggaagagg ataagaagca cgagcggcac cccatcttcg gcaacatcgt ggacgaggtg
      421 gcctaccacg agaagtaccc caccatctac cacctgagaa agaaactggt ggacagcacc
      481 gacaaggccg acctgcggct gatctatctg gccctggccc acatgatcaa gttccggggc
      541 cacttcctga tcgagggcga cctgaacccc gacaacagcg acgtggacaa gctgttcatc
      601 cagctggtgc agacctacaa ccagctgttc gaggaaaacc ccatcaacgc cagcggcgtg
      661 gacgccaagg ccatcctgtc tgccagactg agcaagagca gacggctgga aaatctgatc
      721 gcccagctgc ccggcgagaa gaagaatggc ctgttcggca acctgattgc cctgagcctg
      781 ggcctgaccc ccaacttcaa gagcaacttc gacctggccg aggatgccaa actgcagctg
      841 agcaaggaca cctacgacga cgacctggac aacctgctgg cccagatcgg cgaccagtac
      901 gccgacctgt ttctggccgc caagaacctg tccgacgcca tcctgctgag cgacatcctg
      961 agagtgaaca ccgagatcac caaggccccc ctgagcgcct ctatgatcaa gagatacgac
     1021 gagcaccacc aggacctgac cctgctgaaa gctctcgtgc ggcagcagct gcctgagaag
     1081 tacaaagaga ttttcttcga ccagagcaag aacggctacg ccggctacat cgatggcgga
     1141 gccagccagg aagagttcta caagttcatc aagcccatcc tggaaaagat ggacggcacc
     1201 gaggaactgc tcgtgaagct gaacagagag gacctgctgc ggaagcagcg gaccttcgac
     1261 aacggcagca tcccccacca gatccacctg ggagagctgc acgccattct gcggcggcag
     1321 gaagattttt acccattcct gaaggacaac cgggaaaaga tcgagaagat cctgaccttc
     1381 cgcatcccct actacgtggg ccctctggcc aggggaaaca gcagattcgc ctggatgacc
     1441 agaaagagcg aggaaaccat caccccctgg aacttcgagg aagtggtgga caagggcgcc
     1501 agcgcccaga gcttcatcga gcggatgacc aacttcgata agaacctgcc caacgagaag
     1561 gtgctgccca agcacagcct gctgtacgag tacttcaccg tgtacaacga gctgaccaaa
     1621 gtgaaatacg tgaccgaggg aatgagaaag cccgccttcc tgagcggcga gcagaaaaaa
     1681 gccatcgtgg acctgctgtt caagaccaac cggaaagtga ccgtgaagca gctgaaagag
     1741 gactacttca agaaaatcga gtgcttcgac tccgtggaaa tctccggcgt ggaagatcgg
     1801 ttcaacgcct ccctgggcac ataccacgat ctgctgaaaa ttatcaagga caaggacttc
     1861 ctggacaatg aggaaaacga ggacattctg gaagatatcg tgctgaccct gacactgttt
     1921 gaggacagag agatgatcga ggaacggctg aaaacctatg cccacctgtt cgacgacaaa
     1981 gtgatgaagc agctgaagcg gcggagatac accggctggg gcaggctgag ccggaagctg
     2041 atcaacggca tccgggacaa gcagtccggc aagacaatcc tggatttcct gaagtccgac
     2101 ggcttcgcca acagaaactt catgcagctg atccacgacg acagcctgac ctttaaagag
     2161 gacatccaga aagcccaggt gtccggccag ggcgatagcc tgcacgagca cattgccaat
     2221 ctggccggca gccccgccat taagaagggc atcctgcaga cagtgaaggt ggtggacgag
     2281 ctcgtgaaag tgatgggccg gcacaagccc gagaacatcg tgatcgaaat ggccagagag
     2341 aaccagacca cccagaaggg acagaagaac agccgcgaga gaatgaagcg gatcgaagag
     2401 ggcatcaaag agctgggcag ccagatcctg aaagaacacc ccgtggaaaa cacccagctg
     2461 cagaacgaga agctgtacct gtactacctg cagaatgggc gggatatgta cgtggaccag
     2521 gaactggaca tcaaccggct gtccgactac gatgtggacg ctatcgtgcc tcagagcttt
     2581 ctgaaggacg actccatcga taacaaagtg ctgactcgga gcgacaagaa ccggggcaag
     2641 agcgacaacg tgccctccga agaggtcgtg aagaagatga agaactactg gcgccagctg
     2701 ctgaatgcca agctgattac ccagaggaag ttcgacaatc tgaccaaggc cgagagaggc
     2761 ggcctgagcg aactggataa ggccggcttc atcaagagac agctggtgga aacccggcag
     2821 atcacaaagc acgtggcaca gatcctggac tcccggatga acactaagta cgacgagaac
     2881 gacaaactga tccgggaagt gaaagtgatc accctgaagt ccaagctggt gtccgatttc
     2941 cggaaggatt tccagtttta caaagtgcgc gagatcaaca actaccacca cgcccacgac
     3001 gcctacctga acgccgtcgt gggaaccgcc ctgatcaaaa agtaccctaa gctggaaagc
     3061 gagttcgtgt acggcgacta caaggtgtac gacgtgcgga agatgatcgc caagagcgag
     3121 caggaaatcg gcaaggctac cgccaagtac ttcttctaca gcaacatcat gaactttttc
     3181 aagaccgaga ttaccctggc caacggcgag atccggaagc ggcctctgat cgagacaaac
     3241 ggcgaaacag gcgagatcgt gtgggataag ggccgggact ttgccaccgt gcggaaagtg
     3301 ctgtctatgc cccaagtgaa tatcgtgaaa aagaccgagg tgcagacagg cggcttcagc
     3361 aaagagtcta tcctgcccaa gaggaacagc gacaagctga tcgccagaaa gaaggactgg
     3421 gaccctaaga agtacggcgg cttcgacagc cccaccgtgg cctattctgt gctggtggtg
     3481 gccaaagtgg aaaagggcaa gtccaagaaa ctgaagagtg tgaaagagct gctggggatc
     3541 accatcatgg aaagaagcag cttcgagaag aatcccatcg actttctgga agccaagggc
     3601 tacaaagaag tgaaaaagga cctgatcatc aagctgccta agtactccct gttcgagctg
     3661 gaaaacggcc ggaagagaat gctggcctct gccggcgaac tgcagaaggg aaacgaactg
     3721 gccctgccct ccaaatatgt gaacttcctg tacctggcca gccactatga gaagctgaag
     3781 ggctcccccg aggataatga gcagaaacag ctgtttgtgg aacagcacaa acactacctg
     3841 gacgagatca tcgagcagat cagcgagttc tccaagagag tgatcctggc cgacgctaat
     3901 ctggacaagg tgctgagcgc ctacaacaag cacagagaca agcctatcag agagcaggcc
     3961 gagaatatca tccacctgtt taccctgacc aatctgggag cccctgccgc cttcaagtac
     4021 tttgacacca ccatcgaccg gaagaggtac accagcacca aagaggtgct ggacgccacc
     4081 ctgatccacc agagcatcac cggcctgtac gagacacgga tcgacctgtc tcagctggga
     4141 ggcgacccca agaagaagcg caaggtgcac caccaccacc accacggagg aggtggaagc
     4201 ggaggaggag gaagcggagg aggaggtagc tggcgcgcca tgagcgagct gattaaggag
     4261 aacatgcaca tgaagctgta catggagggc accgtggaca accatcactt caagtgcaca
     4321 tccgagggcg aaggcaagcc ctacgagggc acccagacca tgagaatcaa ggtggtcgag
     4381 ggcggccctc tccccttcgc cttcgacatc ctggctacta gcttcctcta cggcagcaag
     4441 accttcatca accacaccca gggcatcccc gacttcttca agcagtcctt ccctgagggc
     4501 ttcacatggg agagagtcac cacatacgaa gacgggggcg tgctgaccgc tacccaggac
     4561 accagcctcc aggacggctg cctcatctac aacgtcaaga tcagaggggt gaacttcaca
     4621 tccaacggcc ctgtgatgca gaagaaaaca ctcggctggg aggccttcac cgagacgctg
     4681 taccccgctg acggcggcct ggaaggcaga aacgacatgg ccctgaagct cgtgggcggg
     4741 agccatctga tcgcaaacat caagaccaca tatagatcca agaaacccgc taagaacctc
     4801 aagatgcctg gcgtctacta tgtggactac agactggaaa gaatcaagga ggccaacaac
     4861 gagacctacg tcgagcagca cgaggtggca gtggccagat actgcgacct ccctagcaaa
     4921 ctggggcaca agcttaatta gccatggatt aacgactgtg ccttctagtt gccagccatc
     4981 tgttgtttgc ccctcccccg tgccttcctt gaccctggaa ggtgccactc ccactgtcct
     5041 ttcctaataa aatgaggaaa ttgcatcgca ttgtctgagt aggtgtcatt ctattctggg
     5101 gggtggggtg gggcaggaca gcaaggggga ggattgggaa gacaatagca ggcatgctgg
     5161 ggatgcggtg ggctctatgg cctctggcct cgcacacatt ccacatccac cggtaggcgc
     5221 caaccggctc cgttctttgg tggccccttc gcgccacctt ctactcctcc cctagtcagg
     5281 aagttccccc ccgccccgca gctcgcgtcg tgcaggacgt gacaaatgga agtagcacgt
     5341 ctcactagtc tcgtgcagat ggacagcacc gctgagcaat ggaagcgggt aggcctttgg
     5401 ggcagcggcc aatagcagct ttgctccttc gctttctggg ctcagaggct gggaaggggt
     5461 gggtccgggg gcgggctcag gggcgggctc aggggcgggg cgggcgcccg aaggtcctcc
     5521 ggaggcccgg cattctgcac gcttcaaaag cgcacgtctg ccgcgctgtt ctcctcttcc
     5581 tcatctccgg gcctttcgac ctgcaatggc caagcctttg tctcaagaag aatccaccct
     5641 cattgaaaga gcaacggcta caatcaacag catccccatc tctgaagact acagcgtcgc
     5701 cagcgcagct ctctctagcg acggccgcat cttcactggt gtcaatgtat atcattttac
     5761 tgggggacct tgtgcagaac tcgtggtgct gggcactgct gctgctgcgg cagctggcaa
     5821 cctgacttgt atcgtcgcga tcggaaatga gaacaggggc atcttgagcc cctgcggacg
     5881 gtgccgacag gtgcttctcg atctgcatcc tgggatcaaa gccatagtga aggacagtga
     5941 tggacagccg acggcagttg ggattcgtga attgctgccc tctggttatg tgtgggaggg
     6001 ctaaaatcaa cctctggatt acaaaatttg tgaaagattg actggtattc ttaactatgt
     6061 tgctcctttt acgctatgtg gatacgctgc tttaatgcct ttgtatcatg ctattgcttc
     6121 ccgtatggct ttcattttct cctccttgta taaatcctgg ttgctgtctc tttatgagga
     6181 gttgtggccc gttgtcaggc aacgtggcgt ggtgtgcact gtgtttgctg acgcaacccc
     6241 cactggttgg ggcattgcca ccacctgtca gctcctttcc gggactttcg ctttccccct
     6301 ccctattgcc acggcggaac tcatcgccgc ctgccttgcc cgctgctgga caggggctcg
     6361 gctgttgggc actgacaatt ccgtggtgtt gtcggggaaa tcatcgtcct ttccttggct
     6421 gctcgcctgt gttgccacct ggattctgcg cgggacgtcc ttctgctacg tcccttcggc
     6481 cctcaatcca gcggaccttc cttcccgcgg cctgctgccg gctctgcggc ctcttccgcg
     6541 tctccgcctt cgccctcaga cgagtcggat ctccctttgg gccgcctccc cgccggtacc
     6601 taaaagtttt gttactttat agaagaaatt ttgagttttt gttttttttt aataaataaa
     6661 taaacataaa taaattgttt gttgaattta ttattagtat gtaagtgtaa atataataaa
     6721 acttaatatc tattcaaatt aataaataaa cctcgatata cagaccgata aaacacatgc
     6781 gtcaatttta cgcatgatta tctttaacgt acgtcacaat atgattatct ttctagggtt
     6841 aatctagtat taatccagct tttgttccct ttagtgaggg ttaattgcgc gcttggcgta
     6901 atcatggtca tagctgtttc ctgtgtgaaa ttgttatccg ctcacaattc cacacaacat
     6961 acgagccgga agcataaagt gtaaagcctg gggtgcctaa tgagtgagct aactcacatt
     7021 aattgcgttg cgctcactgc ccgctttcca gtcgggaaac ctgtcgtgcc agctgcatta
     7081 atgaatcggc caacgcgcgg ggagaggcgg tttgcgtatt gggcgctctt ccgcttcctc
     7141 gctcactgac tcgctgcgct cggtcgttcg gctgcggcga gcggtatcag ctcactcaaa
     7201 ggcggtaata cggttatcca cagaatcagg ggataacgca ggaaagaaca tgtgagcaaa
     7261 aggccagcaa aaggccagga accgtaaaaa ggccgcgttg ctggcgtttt tccataggct
     7321 ccgcccccct gacgagcatc acaaaaatcg acgctcaagt cagaggtggc gaaacccgac
     7381 aggactataa agataccagg cgtttccccc tggaagctcc ctcgtgcgct ctcctgttcc
     7441 gaccctgccg cttaccggat acctgtccgc ctttctccct tcgggaagcg tggcgctttc
     7501 tcatagctca cgctgtaggt atctcagttc ggtgtaggtc gttcgctcca agctgggctg
     7561 tgtgcacgaa ccccccgttc agcccgaccg ctgcgcctta tccggtaact atcgtcttga
     7621 gtccaacccg gtaagacacg acttatcgcc actggcagca gccactggta acaggattag
     7681 cagagcgagg tatgtaggcg gtgctacaga gttcttgaag tggtggccta actacggcta
     7741 cactagaaga acagtatttg gtatctgcgc tctgctgaag ccagttacct tcggaaaaag
     7801 agttggtagc tcttgatccg gcaaacaaac caccgctggt agcggtggtt tttttgtttg
     7861 caagcagcag attacgcgca gaaaaaaagg atctcaagaa gatcctttga tcttttctac
     7921 ggggtctgac gctcagtgga acgaaaactc acgttaaggg attttggtca tgagattatc
     7981 aaaaaggatc ttcacctaga tccttttaaa ttaaaaatga agttttaaat caatctaaag
     8041 tatatatgag taaacttggt ctgacagtta ccaatgctta atcagtgagg cacctatctc
     8101 agcgatctgt ctatttcgtt catccatagt tgcctgactc cccgtcgtgt agataactac
     8161 gatacgggag ggcttaccat ctggccccag tgctgcaatg ataccgcgag acccacgctc
     8221 accggctcca gatttatcag caataaacca gccagccgga agggccgagc gcagaagtgg
     8281 tcctgcaact ttatccgcct ccatccagtc tattaattgt tgccgggaag ctagagtaag
     8341 tagttcgcca gttaatagtt tgcgcaacgt tgttgccatt gctacaggca tcgtggtgtc
     8401 acgctcgtcg tttggtatgg cttcattcag ctccggttcc caacgatcaa ggcgagttac
     8461 atgatccccc atgttgtgca aaaaagcggt tagctccttc ggtcctccga tcgttgtcag
     8521 aagtaagttg gccgcagtgt tatcactcat ggttatggca gcactgcata attctcttac
     8581 tgtcatgcca tccgtaagat gcttttctgt gactggtgag tactcaacca agtcattctg
     8641 agaatagtgt atgcggcgac cgagttgctc ttgcccggcg tcaatacggg ataataccgc
     8701 gccacatagc agaactttaa aagtgctcat cattggaaaa cgttcttcgg ggcgaaaact
     8761 ctcaaggatc ttaccgctgt tgagatccag ttcgatgtaa cccactcgtg cacccaactg
     8821 atcttcagca tcttttactt tcaccagcgt ttctgggtga gcaaaaacag gaaggcaaaa
     8881 tgccgcaaaa aagggaataa gggcgacacg gaaatgttga atactcatac tcttcctttt
     8941 tcaatattat tgaagcattt atcagggtta ttgtctcatg agcggataca tatttgaatg
     9001 tatttagaaa aataaacaaa taggggttcc gcgcacattt ccccgaaaag tgccacctaa
     9061 attgtaagcg ttaatatttt gttaaaattc gcgttaaatt tttgttaaat cagctcattt
     9121 tttaaccaat aggccgaaat cggcaaaatc ccttataaat caaaagaata gaccgagata
     9181 gggttgagtg ttgttccagt ttggaacaag agtccactat taaagaacgt ggactccaac
     9241 gtcaaagggc gaaaaaccgt ctatcagggc gatggcccac tacgtgaacc atcaccctaa
     9301 tcaagttttt tggggtcgag gtgccgtaaa gcactaaatc ggaaccctaa agggagcccc
     9361 cgatttagag cttgacgggg aaagccggcg aacgtggcga gaaaggaagg gaagaaagcg
     9421 aaaggagcgg gcgctagggc gctggcaagt gtagcggtca cgctgcgcgt aaccaccaca
     9481 cccgccgcgc ttaatgcgcc gctacagggc gcgtcccatt cgccattcag gctgcgcaac
     9541 tgttgggaag ggcgatcggt gcgggcctct tcgctattac gccagctggc gaaaggggga
     9601 tgtgctgcaa ggcgattaag ttgggtaacg ccagggtttt cccagtcacg acgttgtaaa
     9661 acgacggcca gtgagcgcgc gtaatacgac tcactatagg gcgaattggg gctaacccta
     9721 gaaagatagt ctgcgtaaaa ttgacgcatg cattcttgaa atattgctct ctctttctaa
     9781 atagcgcgaa tccgtcgctg tgcatttagg acatctcagt cgccgcttgg agctcccgtg
     9841 aggcgtgctt gtcaatgcgg taagtgtcac tgattttgaa ctataacgac cgcgtgagtc
     9901 aaaatgacgc atgattatct tttacgtgac ttttaagatt taactcatac gataattata
     9961 ttgttatttc atgttctact tacgtgataa cttattatat atatattttc ttgttataga
    10021 tatcaactag aatacgcgta aggatctgcg atcgctccgg tgcccgtcag tgggcagagc
    10081 gcacatcgcc cacagtcccc gagaagttgg ggggaggggt cggcaattga acgggtgcct
    10141 agagaaggtg gcgcggggta aactgggaaa gtgatgtcgt gtactggctc cgcctttttc
    10201 ccgagggtgg gggagaaccg tatataagtg cagtagtcgc cgtgaacgtt ctttttcgca
    10261 acgggtttgc cgccagaaca cagctgaagc ttcgaggggc tcgcatctct ccttcacgcg
    10321 cccgccgccc tacctgaggc cgccatccac gccggttgag tcgcgttctg ccgcctcccg
    10381 cctgtggtgc ctcctgaact gcgtccgccg tctaggtaag tttaaagctc aggtcgagac
    10441 cgggcctttg tccggcgctc ccttggagcc tacctagact cagccggctc tccacgcttt
    10501 gcctgaccct gcttgctcaa ctctacgtct ttgtttcgtt ttctgttctg cgccgttaca
    10561 gatccaagct gtgaccggcg cctactctag agctagcgaa ttcgccgcca ccatgcctaa
    10621 gaaaaagagg aaggtgagcg gccgcggagt tcaggtggaa accatctccc caggagacgg
    10681 gcgcaccttc cccaagcgcg gccagacctg cgtggtgcac tacaccggga tgcttgaaga
    10741 tggaaagaaa gttgattcct cccgggacag aaacaagccc tttaagttta tgctaggcaa
    10801 gcaggaggtg atccgaggct gggaagaagg ggttgcccag atgagtgtgg gtcagagagc
    10861 caaactgact atatctccag attatgccta tggtgccact gggcacccag gcatcatccc
    10921 accacatgcc actctcgtct tcgatgtgga gcttctaaaa ctggaagggg gtggtggaag
    10981 cggaggtgga ggatcaggag gaggaggtag catggatgct aagtcactaa ctgcctggtc
    11041 ccggacactg gtgaccttca aggatgtatt tgtggacttc accagggagg agtggaagct
    11101 gctggacact gctcagcaga tcgtgtacag aaatgtgatg ctggagaact ataagaacct
    11161 ggtttccttg ggttatcagc ttactaagcc agatgtgatc ctccggttgg agaagggaga
    11221 agagccc
//
