LOCUS       sequence-373709-       13480 bp    DNA     circular SYN 15-DEC-2025
DEFINITION  mAID degron-tagged dCas9 fused with tagRFPt, P2A site and tagBFP, 
            under EF1-alpha promoter in a piggyBac plasmid. Expresses 
            Blasticidin resistance, T2A site and osTIR1 under PGK promoter..
ACCESSION   .
VERSION     .
KEYWORDS    .
SOURCE      synthetic DNA construct
  ORGANISM  synthetic DNA construct
REFERENCE   1  (bases 1 to 13480)
  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 13480)
  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: 
            pSLPB3B-mAID-SpdCas9-tagRFPt-P2A-tagBFP-PGK-Blasticidin-T2A-osTIR1
FEATURES             Location/Qualifiers
     source          1..13480
                     /mol_type="other DNA"
                     /organism="synthetic DNA construct"
     primer_bind     412..431
                     /label=Amp-R
                     /note="Ampicillin resistance gene, reverse primer"
     promoter        complement(650..754)
                     /gene="bla"
                     /label=AmpR promoter
     rep_origin      complement(780..1235)
                     /direction=LEFT
                     /label=f1 ori
                     /note="f1 bacteriophage origin of replication; arrow 
                     indicates direction of (+) strand synthesis"
     primer_bind     complement(917..938)
                     /label=F1ori-F
                     /note="F1 origin, forward primer"
     primer_bind     1129..1148
                     /label=F1ori-R
                     /note="F1 origin, reverse primer"
     primer_bind     1362..1384
                     /label=M13/pUC Forward
                     /note="In lacZ gene"
     primer_bind     1376..1393
                     /label=M13 Forward
                     /note="In lacZ gene. Also called M13-F20 or M13 (-21) 
                     Forward"
     primer_bind     1377..1393
                     /label=M13 fwd
                     /note="common sequencing primer, one of multiple similar 
                     variants"
     primer_bind     1403..1422
                     /label=T7
                     /note="T7 promoter, forward primer"
     promoter        1403..1421
                     /label=T7 promoter
                     /note="promoter for bacteriophage T7 RNA polymerase"
     repeat_region   1437..1471
                     /label=piggyBac left (5') inverted repeat
                     /note="piggyBac transposon-specific inverted terminal 
                     repeat sequence (ITR)"
     promoter        1793..2004
                     /label=EF-1-alpha core promoter
                     /note="core promoter for human elongation factor 
                     EF-1-alpha"
     LTR             2017..2285
                     /label=5' LTR (truncated)
                     /note="truncated 5' long terminal repeat (LTR) from human 
                     T-cell leukemia virus (HTLV) type 1"
     CDS             2337..2357
                     /codon_start=1
                     /product="nuclear localization signal of SV40 (simian virus
                     40) large T antigen"
                     /label=SV40 NLS
                     /translation="PKKKRKV"
     CDS             2367..2567
                     /codon_start=1
                     /product="minimal degron from the auxin-responsive IAA17 
                     protein of Arabidopsis thaliana (Kubota et al., 2013)"
                     /label=mini-AID
                     /note="acts as an auxin-inducible degron (AID) in the 
                     presence of TIR1"
                     /translation="EKSACPKDPAKPPAKAQVVGWPPVRSYRKNVMVSCQKSSGGPEAA
                     AFVKVSMDGAPYLRKIDLRMYK"
     CDS             join(2619..2642,2643..2645,2646..5132,5133..5135,
                     5136..6719)
                     /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             2619..6719
                     /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             6720..6740
                     /codon_start=1
                     /product="nuclear localization signal of SV40 (simian virus
                     40) large T antigen"
                     /label=SV40 NLS
                     /translation="PKKKRKV"
     CDS             6741..6758
                     /codon_start=1
                     /product="6xHis affinity tag"
                     /label=6xHis
                     /translation="HHHHHH"
     CDS             6813..7541
                     /codon_start=1
                     /product="photostable variant of TagRFP (Shaner et al., 
                     2008)"
                     /label=TagRFP-T
                     /translation="VSKGEELIKENMHMKLYMEGTVNNHHFKCTSEGEGKPYEGTQTMR
                     IKVVEGGPLPFAFDILATSFMYGSRTFINHTQGIPDFFKQSFPEGFTWERVTTYEDGGV
                     LTATQDTSLQDGCLIYNVKIRGVNFPSNGPVMQKKTLGWEANTEMLYPADGGLEGRTDM
                     ALKLVGGGHLICNFKTTYRSKKPAKNLKMPGVYYVDHRLERIKEADKETYVEQHEVAVA
                     RYCDLPSKLGHKLNGMDELYK"
     CDS             7560..7616
                     /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             7617..8318
                     /codon_start=1
                     /product="monomeric blue fluorescent protein"
                     /label=TagBFP
                     /note="mammalian codon-optimized"
                     /translation="MSELIKENMHMKLYMEGTVDNHHFKCTSEGEGKPYEGTQTMRIKV
                     VEGGPLPFAFDILATSFLYGSKTFINHTQGIPDFFKQSFPEGFTWERVTTYEDGGVLTA
                     TQDTSLQDGCLIYNVKIRGVNFTSNGPVMQKKTLGWEAFTETLYPADGGLEGRNDMALK
                     LVGGSHLIANIKTTYRSKKPAKNLKMPGVYYVDYRLERIKEANNETYVEQHEVAVARYC
                     DLPSKLGHKLN"
     polyA_signal    8333..8557
                     /label=bGH poly(A) signal
                     /note="bovine growth hormone polyadenylation signal"
     primer_bind     8908..8927
                     /label=mPGK-F
                     /note="Mouse PGK promoter, forward primer"
     CDS             8983..9378
                     /codon_start=1
                     /gene="Aspergillus terreus BSD"
                     /product="blasticidin S deaminase"
                     /label=BSD
                     /note="confers resistance to blasticidin"
                     /translation="MAKPLSQEESTLIERATATINSIPISEDYSVASAALSSDGRIFTG
                     VNVYHFTGGPCAELVVLGTAAAAAAGNLTCIVAIGNENRGILSPCGRCRQVLLDLHPGI
                     KAIVKDSDGQPTAVGIRELLPSGYVWEG"
     CDS             9388..9441
                     /codon_start=1
                     /product="2A peptide from Thosea asigna virus capsid 
                     protein"
                     /label=T2A
                     /note="Eukaryotic ribosomes fail to insert a peptide bond 
                     between the Gly and Pro residues, yielding separate 
                     polypeptides."
                     /translation="EGRGSLLTCGDVEENPGP"
     CDS             9442..11163
                     /codon_start=1
                     /product="rice transport inhibitor response 1 protein"
                     /label=OsTIR1
                     /note="F-box protein that mediates auxin-dependent 
                     interaction of a ubiquitin ligase with an auxin-inducible 
                     degron (Tan et al., 2007)"
                     /translation="TYFPEEVVEHIFSFLPAQRDRNTVSLVCKVWYEIERLSRRGVFVG
                     NCYAVRAGRVAARFPNVRALTVKGKPHFADFNLVPPDWGGYAGPWIEAAARGCHGLEEL
                     RMKRMVVSDESLELLARSFPRFRALVLISCEGFSTDGLAAVASHCKLLRELDLQENEVE
                     DRGPRWLSCFPDSCTSLVSLNFACIKGEVNAGSLERLVSRSPNLRSLRLNRSVSVDTLA
                     KILLRTPNLEDLGTGNLTDDFQTESYFKLTSALEKCKMLRSLSGFWDASPVCLSFIYPL
                     CAQLTGLNLSYAPTLDASDLTKMISRCVKLQRLWVLDCISDKGLQVVASSCKDLQELRV
                     FPSDFYVAGYSAVTEEGLVAVSLGCPKLNSLLYFCHQMTNAALVTVAKNCPNFTRFRLC
                     ILEPGKPDVVTSQPLDEGFGAIVRECKGLQRLSISGLLTDKVFMYIGKYAKQLEMLSIA
                     FAGDSDKGMMHVMNGCKNLRKLEIRDSPFGDAALLGNFARYETMRSLWMSSCNVTLKGC
                     QVLASKMPMLNVEVINERDGSNEMEENHGDLPKVEKLYVYRTTAGARDDAPNFVKIL"
     CDS             11173..11202
                     /codon_start=1
                     /product="Myc (human c-Myc proto-oncogene) epitope tag"
                     /label=Myc
                     /translation="EQKLISEEDL"
     misc_feature    11206..11794
                     /label=WPRE
                     /note="woodchuck hepatitis virus posttranscriptional 
                     regulatory element"
     primer_bind     complement(11259..11279)
                     /label=WPRE-R
                     /note="WPRE, reverse primer"
     CDS             complement(11677..11688)
                     /codon_start=1
                     /product="Factor Xa recognition and cleavage site"
                     /label=Factor Xa site
                     /translation="IEGR"
     repeat_region   11977..12039
                     /label=piggyBac right (3') inverted repeat
                     /note="piggyBac transposon-specific inverted terminal 
                     repeat sequence (ITR)"
     primer_bind     complement(12069..12089)
                     /label=T3
                     /note="T3 promoter, forward primer"
     promoter        complement(12069..12087)
                     /label=T3 promoter
                     /note="promoter for bacteriophage T3 RNA polymerase"
     primer_bind     complement(12108..12124)
                     /label=M13 rev
                     /note="common sequencing primer, one of multiple similar 
                     variants"
     primer_bind     complement(12108..12124)
                     /label=M13 Reverse
                     /note="In lacZ gene. Also called M13-rev"
     primer_bind     complement(12121..12143)
                     /label=M13/pUC Reverse
                     /note="In lacZ gene"
     protein_bind    12132..12148
                     /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(12156..12162,12163..12180,12181..12186))
                     /label=lac promoter
                     /note="promoter for the E. coli lac operon"
     protein_bind    12201..12222
                     /label=CAP binding site
                     /bound_moiety="E. coli catabolite activator protein"
                     /note="CAP binding activates transcription in the presence 
                     of cAMP."
     primer_bind     complement(12339..12356)
                     /label=L4440
                     /note="L4440 vector, forward primer"
     rep_origin      complement(12510..13098)
                     /direction=LEFT
                     /label=ori
                     /note="high-copy-number ColE1/pMB1/pBR322/pUC origin of 
                     replication"
     primer_bind     complement(12590..12609)
                     /label=pBR322ori-F
                     /note="pBR322 origin, forward primer"
     CDS             complement(join(13269..13480,1..580,581..649))
                     /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"
ORIGIN
        1 gtcctgcaac tttatccgcc tccatccagt ctattaattg ttgccgggaa gctagagtaa
       61 gtagttcgcc agttaatagt ttgcgcaacg ttgttgccat tgctacaggc atcgtggtgt
      121 cacgctcgtc gtttggtatg gcttcattca gctccggttc ccaacgatca aggcgagtta
      181 catgatcccc catgttgtgc aaaaaagcgg ttagctcctt cggtcctccg atcgttgtca
      241 gaagtaagtt ggccgcagtg ttatcactca tggttatggc agcactgcat aattctctta
      301 ctgtcatgcc atccgtaaga tgcttttctg tgactggtga gtactcaacc aagtcattct
      361 gagaatagtg tatgcggcga ccgagttgct cttgcccggc gtcaatacgg gataataccg
      421 cgccacatag cagaacttta aaagtgctca tcattggaaa acgttcttcg gggcgaaaac
      481 tctcaaggat cttaccgctg ttgagatcca gttcgatgta acccactcgt gcacccaact
      541 gatcttcagc atcttttact ttcaccagcg tttctgggtg agcaaaaaca ggaaggcaaa
      601 atgccgcaaa aaagggaata agggcgacac ggaaatgttg aatactcata ctcttccttt
      661 ttcaatatta ttgaagcatt tatcagggtt attgtctcat gagcggatac atatttgaat
      721 gtatttagaa aaataaacaa ataggggttc cgcgcacatt tccccgaaaa gtgccaccta
      781 aattgtaagc gttaatattt tgttaaaatt cgcgttaaat ttttgttaaa tcagctcatt
      841 ttttaaccaa taggccgaaa tcggcaaaat cccttataaa tcaaaagaat agaccgagat
      901 agggttgagt gttgttccag tttggaacaa gagtccacta ttaaagaacg tggactccaa
      961 cgtcaaaggg cgaaaaaccg tctatcaggg cgatggccca ctacgtgaac catcacccta
     1021 atcaagtttt ttggggtcga ggtgccgtaa agcactaaat cggaacccta aagggagccc
     1081 ccgatttaga gcttgacggg gaaagccggc gaacgtggcg agaaaggaag ggaagaaagc
     1141 gaaaggagcg ggcgctaggg cgctggcaag tgtagcggtc acgctgcgcg taaccaccac
     1201 acccgccgcg cttaatgcgc cgctacaggg cgcgtcccat tcgccattca ggctgcgcaa
     1261 ctgttgggaa gggcgatcgg tgcgggcctc ttcgctatta cgccagctgg cgaaaggggg
     1321 atgtgctgca aggcgattaa gttgggtaac gccagggttt tcccagtcac gacgttgtaa
     1381 aacgacggcc agtgagcgcg cgtaatacga ctcactatag ggcgaattgg ggctaaccct
     1441 agaaagatag tctgcgtaaa attgacgcat gcattcttga aatattgctc tctctttcta
     1501 aatagcgcga atccgtcgct gtgcatttag gacatctcag tcgccgcttg gagctcccgt
     1561 gaggcgtgct tgtcaatgcg gtaagtgtca ctgattttga actataacga ccgcgtgagt
     1621 caaaatgacg catgattatc ttttacgtga cttttaagat ttaactcata cgataattat
     1681 attgttattt catgttctac ttacgtgata acttattata tatatatttt cttgttatag
     1741 atatcaacta gaatacgcgt aaggatctgc gatcgctccg gtgcccgtca gtgggcagag
     1801 cgcacatcgc ccacagtccc cgagaagttg gggggagggg tcggcaattg aacgggtgcc
     1861 tagagaaggt ggcgcggggt aaactgggaa agtgatgtcg tgtactggct ccgccttttt
     1921 cccgagggtg ggggagaacc gtatataagt gcagtagtcg ccgtgaacgt tctttttcgc
     1981 aacgggtttg ccgccagaac acagctgaag cttcgagggg ctcgcatctc tccttcacgc
     2041 gcccgccgcc ctacctgagg ccgccatcca cgccggttga gtcgcgttct gccgcctccc
     2101 gcctgtggtg cctcctgaac tgcgtccgcc gtctaggtaa gtttaaagct caggtcgaga
     2161 ccgggccttt gtccggcgct cccttggagc ctacctagac tcagccggct ctccacgctt
     2221 tgcctgaccc tgcttgctca actctacgtc tttgtttcgt tttctgttct gcgccgttac
     2281 agatccaagc tgtgaccggc gcctactcta gagctagcga attcgccgcc accatgccta
     2341 agaaaaagag gaaggtgagc ggccgcgaga agagtgcttg tcctaaagat ccagccaaac
     2401 ctccggccaa ggcacaagtt gtgggatggc caccggtgag atcataccgg aagaacgtga
     2461 tggtttcctg ccaaaaatca agcggtggcc cggaggcggc ggcgttcgtg aaggtatcaa
     2521 tggacggagc accgtacttg aggaaaatcg atttgaggat gtataaagga tccggcggat
     2581 ctggaggtag tggtggatct ggaggttctg gtggctccga caagaagtac agcatcggcc
     2641 tggccatcgg caccaactct gtgggctggg ccgtgatcac cgacgagtac aaggtgccca
     2701 gcaagaaatt caaggtgctg ggcaacaccg accggcacag catcaagaag aacctgatcg
     2761 gcgccctgct gttcgacagc ggagaaacag ccgaggccac ccggctgaag agaaccgcca
     2821 gaagaagata caccagacgg aagaaccgga tctgctatct gcaagagatc ttcagcaacg
     2881 agatggccaa ggtggacgac agcttcttcc acagactgga agagtccttc ctggtggaag
     2941 aggataagaa gcacgagcgg caccccatct tcggcaacat cgtggacgag gtggcctacc
     3001 acgagaagta ccccaccatc taccacctga gaaagaaact ggtggacagc accgacaagg
     3061 ccgacctgcg gctgatctat ctggccctgg cccacatgat caagttccgg ggccacttcc
     3121 tgatcgaggg cgacctgaac cccgacaaca gcgacgtgga caagctgttc atccagctgg
     3181 tgcagaccta caaccagctg ttcgaggaaa accccatcaa cgccagcggc gtggacgcca
     3241 aggccatcct gtctgccaga ctgagcaaga gcagacggct ggaaaatctg atcgcccagc
     3301 tgcccggcga gaagaagaat ggcctgttcg gcaacctgat tgccctgagc ctgggcctga
     3361 cccccaactt caagagcaac ttcgacctgg ccgaggatgc caaactgcag ctgagcaagg
     3421 acacctacga cgacgacctg gacaacctgc tggcccagat cggcgaccag tacgccgacc
     3481 tgtttctggc cgccaagaac ctgtccgacg ccatcctgct gagcgacatc ctgagagtga
     3541 acaccgagat caccaaggcc cccctgagcg cctctatgat caagagatac gacgagcacc
     3601 accaggacct gaccctgctg aaagctctcg tgcggcagca gctgcctgag aagtacaaag
     3661 agattttctt cgaccagagc aagaacggct acgccggcta catcgatggc ggagccagcc
     3721 aggaagagtt ctacaagttc atcaagccca tcctggaaaa gatggacggc accgaggaac
     3781 tgctcgtgaa gctgaacaga gaggacctgc tgcggaagca gcggaccttc gacaacggca
     3841 gcatccccca ccagatccac ctgggagagc tgcacgccat tctgcggcgg caggaagatt
     3901 tttacccatt cctgaaggac aaccgggaaa agatcgagaa gatcctgacc ttccgcatcc
     3961 cctactacgt gggccctctg gccaggggaa acagcagatt cgcctggatg accagaaaga
     4021 gcgaggaaac catcaccccc tggaacttcg aggaagtggt ggacaagggc gccagcgccc
     4081 agagcttcat cgagcggatg accaacttcg ataagaacct gcccaacgag aaggtgctgc
     4141 ccaagcacag cctgctgtac gagtacttca ccgtgtacaa cgagctgacc aaagtgaaat
     4201 acgtgaccga gggaatgaga aagcccgcct tcctgagcgg cgagcagaaa aaagccatcg
     4261 tggacctgct gttcaagacc aaccggaaag tgaccgtgaa gcagctgaaa gaggactact
     4321 tcaagaaaat cgagtgcttc gactccgtgg aaatctccgg cgtggaagat cggttcaacg
     4381 cctccctggg cacataccac gatctgctga aaattatcaa ggacaaggac ttcctggaca
     4441 atgaggaaaa cgaggacatt ctggaagata tcgtgctgac cctgacactg tttgaggaca
     4501 gagagatgat cgaggaacgg ctgaaaacct atgcccacct gttcgacgac aaagtgatga
     4561 agcagctgaa gcggcggaga tacaccggct ggggcaggct gagccggaag ctgatcaacg
     4621 gcatccggga caagcagtcc ggcaagacaa tcctggattt cctgaagtcc gacggcttcg
     4681 ccaacagaaa cttcatgcag ctgatccacg acgacagcct gacctttaaa gaggacatcc
     4741 agaaagccca ggtgtccggc cagggcgata gcctgcacga gcacattgcc aatctggccg
     4801 gcagccccgc cattaagaag ggcatcctgc agacagtgaa ggtggtggac gagctcgtga
     4861 aagtgatggg ccggcacaag cccgagaaca tcgtgatcga aatggccaga gagaaccaga
     4921 ccacccagaa gggacagaag aacagccgcg agagaatgaa gcggatcgaa gagggcatca
     4981 aagagctggg cagccagatc ctgaaagaac accccgtgga aaacacccag ctgcagaacg
     5041 agaagctgta cctgtactac ctgcagaatg ggcgggatat gtacgtggac caggaactgg
     5101 acatcaaccg gctgtccgac tacgatgtgg acgctatcgt gcctcagagc tttctgaagg
     5161 acgactccat cgataacaaa gtgctgactc ggagcgacaa gaaccggggc aagagcgaca
     5221 acgtgccctc cgaagaggtc gtgaagaaga tgaagaacta ctggcgccag ctgctgaatg
     5281 ccaagctgat tacccagagg aagttcgaca atctgaccaa ggccgagaga ggcggcctga
     5341 gcgaactgga taaggccggc ttcatcaaga gacagctggt ggaaacccgg cagatcacaa
     5401 agcacgtggc acagatcctg gactcccgga tgaacactaa gtacgacgag aacgacaaac
     5461 tgatccggga agtgaaagtg atcaccctga agtccaagct ggtgtccgat ttccggaagg
     5521 atttccagtt ttacaaagtg cgcgagatca acaactacca ccacgcccac gacgcctacc
     5581 tgaacgccgt cgtgggaacc gccctgatca aaaagtaccc taagctggaa agcgagttcg
     5641 tgtacggcga ctacaaggtg tacgacgtgc ggaagatgat cgccaagagc gagcaggaaa
     5701 tcggcaaggc taccgccaag tacttcttct acagcaacat catgaacttt ttcaagaccg
     5761 agattaccct ggccaacggc gagatccgga agcggcctct gatcgagaca aacggcgaaa
     5821 caggcgagat cgtgtgggat aagggccggg actttgccac cgtgcggaaa gtgctgtcta
     5881 tgccccaagt gaatatcgtg aaaaagaccg aggtgcagac aggcggcttc agcaaagagt
     5941 ctatcctgcc caagaggaac agcgacaagc tgatcgccag aaagaaggac tgggacccta
     6001 agaagtacgg cggcttcgac agccccaccg tggcctattc tgtgctggtg gtggccaaag
     6061 tggaaaaggg caagtccaag aaactgaaga gtgtgaaaga gctgctgggg atcaccatca
     6121 tggaaagaag cagcttcgag aagaatccca tcgactttct ggaagccaag ggctacaaag
     6181 aagtgaaaaa ggacctgatc atcaagctgc ctaagtactc cctgttcgag ctggaaaacg
     6241 gccggaagag aatgctggcc tctgccggcg aactgcagaa gggaaacgaa ctggccctgc
     6301 cctccaaata tgtgaacttc ctgtacctgg ccagccacta tgagaagctg aagggctccc
     6361 ccgaggataa tgagcagaaa cagctgtttg tggaacagca caaacactac ctggacgaga
     6421 tcatcgagca gatcagcgag ttctccaaga gagtgatcct ggccgacgct aatctggaca
     6481 aggtgctgag cgcctacaac aagcacagag acaagcctat cagagagcag gccgagaata
     6541 tcatccacct gtttaccctg accaatctgg gagcccctgc cgccttcaag tactttgaca
     6601 ccaccatcga ccggaagagg tacaccagca ccaaagaggt gctggacgcc accctgatcc
     6661 accagagcat caccggcctg tacgagacac ggatcgacct gtctcagctg ggaggcgacc
     6721 ccaagaagaa gcgcaaggtg caccaccacc accaccacgg aggaggtgga agcggaggag
     6781 gaggaagcgg aggaggaggt agctggcgcg ccgtgtctaa gggcgaagag ctgattaagg
     6841 agaacatgca catgaagctg tacatggagg gcaccgtgaa caaccaccac ttcaagtgca
     6901 catccgaggg cgaaggcaag ccctacgagg gcacccagac catgagaatc aaggtggtcg
     6961 agggcggccc tctccccttc gccttcgaca tcctggctac cagcttcatg tacggcagca
     7021 gaaccttcat caaccacacc cagggcatcc ccgacttctt taagcagtcc ttccctgagg
     7081 gcttcacatg ggagagagtc accacatacg aagacggggg cgtgctgacc gctacccagg
     7141 acaccagcct ccaggacggc tgcctcatct acaacgtcaa gatcagaggg gtgaacttcc
     7201 catccaacgg ccctgtgatg cagaagaaaa cactcggctg ggaggccaac accgagatgc
     7261 tgtaccccgc tgacggcggc ctggaaggca gaaccgacat ggccctgaag ctcgtgggcg
     7321 ggggccacct gatctgcaac ttcaagacca catacagatc caagaaaccc gctaagaacc
     7381 tcaagatgcc cggcgtctac tatgtggacc acagactgga aagaatcaag gaggccgaca
     7441 aagagaccta cgtcgagcag cacgaggtgg ctgtggccag atactgcgac ctccctagca
     7501 aactggggca caaacttaat ggcatggacg agctgtacaa gttaattaac ggaagcggag
     7561 ctactaactt cagcctgctg aagcaggctg gagacgtgga ggagaaccct ggacctatga
     7621 gcgagctgat taaggagaac atgcacatga agctgtacat ggagggcacc gtggacaacc
     7681 atcacttcaa gtgcacatcc gagggcgaag gcaagcccta cgagggcacc cagaccatga
     7741 gaatcaaggt ggtcgagggc ggccctctcc ccttcgcctt cgacatcctg gctactagct
     7801 tcctctacgg cagcaagacc ttcatcaacc acacccaggg catccccgac ttcttcaagc
     7861 agtccttccc tgagggcttc acatgggaga gagtcaccac atacgaagac gggggcgtgc
     7921 tgaccgctac ccaggacacc agcctccagg acggctgcct catctacaac gtcaagatca
     7981 gaggggtgaa cttcacatcc aacggccctg tgatgcagaa gaaaacactc ggctgggagg
     8041 ccttcaccga gacgctgtac cccgctgacg gcggcctgga aggcagaaac gacatggccc
     8101 tgaagctcgt gggcgggagc catctgatcg caaacatcaa gaccacatat agatccaaga
     8161 aacccgctaa gaacctcaag atgcctggcg tctactatgt ggactacaga ctggaaagaa
     8221 tcaaggaggc caacaacgag acctacgtcg agcagcacga ggtggcagtg gccagatact
     8281 gcgacctccc tagcaaactg gggcacaagc ttaattagac gcgtattaac gactgtgcct
     8341 tctagttgcc agccatctgt tgtttgcccc tcccccgtgc cttccttgac cctggaaggt
     8401 gccactccca ctgtcctttc ctaataaaat gaggaaattg catcgcattg tctgagtagg
     8461 tgtcattcta ttctgggggg tggggtgggg caggacagca agggggagga ttgggaagac
     8521 aatagcaggc atgctgggga tgcggtgggc tctatggcct ctggcctcgc acacattcca
     8581 catccaccgg taggcgccaa ccggctccgt tctttggtgg ccccttcgcg ccaccttcta
     8641 ctcctcccct agtcaggaag ttcccccccg ccccgcagct cgcgtcgtgc aggacgtgac
     8701 aaatggaagt agcacgtctc actagtctcg tgcagatgga cagcaccgct gagcaatgga
     8761 agcgggtagg cctttggggc agcggccaat agcagctttg ctccttcgct ttctgggctc
     8821 agaggctggg aaggggtggg tccgggggcg ggctcagggg cgggctcagg ggcggggcgg
     8881 gcgcccgaag gtcctccgga ggcccggcat tctgcacgct tcaaaagcgc acgtctgccg
     8941 cgctgttctc ctcttcctca tctccgggcc tttcgacctg caatggccaa gcctttgtct
     9001 caagaagaat ccaccctcat tgaaagagca acggctacaa tcaacagcat ccccatctct
     9061 gaagactaca gcgtcgccag cgcagctctc tctagcgacg gccgcatctt cactggtgtc
     9121 aatgtatatc attttactgg gggaccttgt gcagaactcg tggtgctggg cactgctgct
     9181 gctgcggcag ctggcaacct gacttgtatc gtcgcgatcg gaaatgagaa caggggcatc
     9241 ttgagcccct gcggacggtg ccgacaggtg cttctcgatc tgcatcctgg gatcaaagcc
     9301 atagtgaagg acagtgatgg acagccgacg gcagttggga ttcgtgaatt gctgccctct
     9361 ggttatgtgt gggagggcgg aagcggagag ggcagaggaa gtcttctaac atgcggtgac
     9421 gtggaggaga atcccggccc tacatacttt cctgaagagg tcgtcgaaca catttttagc
     9481 ttcctgcctg cacagagaga tagaaacaca gtgagcctgg tctgcaaagt gtggtacgag
     9541 atcgaacgcc tgagccggag aggagtgttc gtcggcaact gctatgctgt gagagcaggc
     9601 agggtcgccg ctaggtttcc aaatgtgcgc gcactgaccg tcaaggggaa accccacttc
     9661 gccgacttta acctggtgcc ccctgattgg ggaggatacg ccggcccttg gatcgaggca
     9721 gccgctcgcg gctgtcatgg actggaggaa ctgcgcatga agcgaatggt ggtctctgac
     9781 gaaagtctgg agctgctggc tcggagcttc cctaggtttc gcgcactggt gctgatttct
     9841 tgcgaaggct tcagcaccga tggactggca gccgtggcct cccactgtaa gctgctgcgg
     9901 gagctggacc tccaggagaa tgaagtggag gatagaggcc ccagatggct gtcttgcttc
     9961 ccagactcat gtaccagcct ggtgtccctg aactttgcct gcatcaaagg cgaagtgaat
    10021 gctgggtccc tggagcggct ggtctcaaga agccccaacc tgaggtctct gcggctgaac
    10081 cggagcgtga gcgtggacac tctggctaag attctgctga gaacccctaa cctggaggat
    10141 ctgggaaccg gcaatctgac agacgatttc cagacagaat cctactttaa actgacttct
    10201 gccctggaga agtgtaaaat gctgaggagt ctgtcaggat tctgggatgc ttcacccgtg
    10261 tgcctgagct ttatctaccc tctgtgtgca cagctgacag gcctgaacct gagctatgca
    10321 ccaaccctgg acgccagtga tctgacaaag atgatctcac gctgcgtgaa actccagcga
    10381 ctgtgggtgc tggactgtat ttccgataag gggctccagg tggtcgccag ctcctgcaag
    10441 gacctccagg agctgagagt gttcccatct gatttttacg tggccggata tagtgctgtc
    10501 actgaggaag gcctggtggc agtctcactg ggatgcccaa agctgaacag cctgctgtat
    10561 ttctgtcatc agatgactaa tgctgcactg gtgaccgtcg ccaagaactg ccctaatttc
    10621 acccgatttc ggctgtgtat tctggaacca ggcaaacccg acgtggtcac atcccagcca
    10681 ctggatgaag ggtttggagc tatcgtgaga gagtgcaagg gactccagag gctgagcatt
    10741 tccggcctgc tgacagacaa agtgttcatg tacatcggca agtatgctaa gcagctggag
    10801 atgctgagca ttgcatttgc cggagactcc gataagggca tgatgcacgt gatgaacggg
    10861 tgtaagaatc tgcgaaaact ggaaatccgg gacagccctt tcggggatgc cgctctgctg
    10921 ggaaactttg ccagatacga gacaatgagg agcctgtgga tgtctagttg caatgtgact
    10981 ctgaagggct gtcaggtcct ggctagtaaa atgcctatgc tgaacgtgga agtcattaat
    11041 gagcgggacg ggtctaacga aatggaggaa aatcatggcg acctgccaaa ggtggagaaa
    11101 ctgtatgtgt atcggaccac cgcaggggca agagatgatg ctcccaactt tgtgaagatt
    11161 ctgggaagcg gagaacaaaa actcatctca gaagaggatc tgtgaaatca acctctggat
    11221 tacaaaattt gtgaaagatt gactggtatt cttaactatg ttgctccttt tacgctatgt
    11281 ggatacgctg ctttaatgcc tttgtatcat gctattgctt cccgtatggc tttcattttc
    11341 tcctccttgt ataaatcctg gttgctgtct ctttatgagg agttgtggcc cgttgtcagg
    11401 caacgtggcg tggtgtgcac tgtgtttgct gacgcaaccc ccactggttg gggcattgcc
    11461 accacctgtc agctcctttc cgggactttc gctttccccc tccctattgc cacggcggaa
    11521 ctcatcgccg cctgccttgc ccgctgctgg acaggggctc ggctgttggg cactgacaat
    11581 tccgtggtgt tgtcggggaa atcatcgtcc tttccttggc tgctcgcctg tgttgccacc
    11641 tggattctgc gcgggacgtc cttctgctac gtcccttcgg ccctcaatcc agcggacctt
    11701 ccttcccgcg gcctgctgcc ggctctgcgg cctcttccgc gtctccgcct tcgccctcag
    11761 acgagtcgga tctccctttg ggccgcctcc ccgccggtac ctaaaagttt tgttacttta
    11821 tagaagaaat tttgagtttt tgtttttttt taataaataa ataaacataa ataaattgtt
    11881 tgttgaattt attattagta tgtaagtgta aatataataa aacttaatat ctattcaaat
    11941 taataaataa acctcgatat acagaccgat aaaacacatg cgtcaatttt acgcatgatt
    12001 atctttaacg tacgtcacaa tatgattatc tttctagggt taatctagta ttaatccagc
    12061 ttttgttccc tttagtgagg gttaattgcg cgcttggcgt aatcatggtc atagctgttt
    12121 cctgtgtgaa attgttatcc gctcacaatt ccacacaaca tacgagccgg aagcataaag
    12181 tgtaaagcct ggggtgccta atgagtgagc taactcacat taattgcgtt gcgctcactg
    12241 cccgctttcc agtcgggaaa cctgtcgtgc cagctgcatt aatgaatcgg ccaacgcgcg
    12301 gggagaggcg gtttgcgtat tgggcgctct tccgcttcct cgctcactga ctcgctgcgc
    12361 tcggtcgttc ggctgcggcg agcggtatca gctcactcaa aggcggtaat acggttatcc
    12421 acagaatcag gggataacgc aggaaagaac atgtgagcaa aaggccagca aaaggccagg
    12481 aaccgtaaaa aggccgcgtt gctggcgttt ttccataggc tccgcccccc tgacgagcat
    12541 cacaaaaatc gacgctcaag tcagaggtgg cgaaacccga caggactata aagataccag
    12601 gcgtttcccc ctggaagctc cctcgtgcgc tctcctgttc cgaccctgcc gcttaccgga
    12661 tacctgtccg cctttctccc ttcgggaagc gtggcgcttt ctcatagctc acgctgtagg
    12721 tatctcagtt cggtgtaggt cgttcgctcc aagctgggct gtgtgcacga accccccgtt
    12781 cagcccgacc gctgcgcctt atccggtaac tatcgtcttg agtccaaccc ggtaagacac
    12841 gacttatcgc cactggcagc agccactggt aacaggatta gcagagcgag gtatgtaggc
    12901 ggtgctacag agttcttgaa gtggtggcct aactacggct acactagaag aacagtattt
    12961 ggtatctgcg ctctgctgaa gccagttacc ttcggaaaaa gagttggtag ctcttgatcc
    13021 ggcaaacaaa ccaccgctgg tagcggtggt ttttttgttt gcaagcagca gattacgcgc
    13081 agaaaaaaag gatctcaaga agatcctttg atcttttcta cggggtctga cgctcagtgg
    13141 aacgaaaact cacgttaagg gattttggtc atgagattat caaaaaggat cttcacctag
    13201 atccttttaa attaaaaatg aagttttaaa tcaatctaaa gtatatatga gtaaacttgg
    13261 tctgacagtt accaatgctt aatcagtgag gcacctatct cagcgatctg tctatttcgt
    13321 tcatccatag ttgcctgact ccccgtcgtg tagataacta cgatacggga gggcttacca
    13381 tctggcccca gtgctgcaat gataccgcga gacccacgct caccggctcc agatttatca
    13441 gcaataaacc agccagccgg aagggccgag cgcagaagtg
//
