LOCUS       sequence-373501-       11929 bp    DNA     circular SYN 15-DEC-2025
DEFINITION  ecDHFR degron-tagged dCas9 fused with tagRFPt, P2A site and 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 11929)
  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 11929)
  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-ecDHFR-SpdCas9-tagRFPt-P2A-tagBFP-PGK-Blasticidin
FEATURES             Location/Qualifiers
     source          1..11929
                     /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..2840
                     /codon_start=1
                     /gene="folA"
                     /product="E. coli dihydrofolate reductase"
                     /label=ecDHFR
                     /translation="ISLIAALAVDYVIGMENAMPWNLPADLAWFKRNTLNKPVIMGRHT
                     WESIGRPLPGRKNIILSSQPSTDDRVTWVKSVDEAIAACGDVPEIMVIGGGRVIEQFLP
                     KAQKLYLTHIDAEVEGDTHFPDYEPDDWESVFSEFHDADAQNSHSYCFEILERR"
     CDS             join(2892..2915,2916..2918,2919..5405,5406..5408,
                     5409..6992)
                     /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             2892..6992
                     /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             6993..7013
                     /codon_start=1
                     /product="nuclear localization signal of SV40 (simian virus
                     40) large T antigen"
                     /label=SV40 NLS
                     /translation="PKKKRKV"
     CDS             7014..7031
                     /codon_start=1
                     /product="6xHis affinity tag"
                     /label=6xHis
                     /translation="HHHHHH"
     CDS             7086..7814
                     /codon_start=1
                     /product="photostable variant of TagRFP (Shaner et al., 
                     2008)"
                     /label=TagRFP-T
                     /translation="VSKGEELIKENMHMKLYMEGTVNNHHFKCTSEGEGKPYEGTQTMR
                     IKVVEGGPLPFAFDILATSFMYGSRTFINHTQGIPDFFKQSFPEGFTWERVTTYEDGGV
                     LTATQDTSLQDGCLIYNVKIRGVNFPSNGPVMQKKTLGWEANTEMLYPADGGLEGRTDM
                     ALKLVGGGHLICNFKTTYRSKKPAKNLKMPGVYYVDHRLERIKEADKETYVEQHEVAVA
                     RYCDLPSKLGHKLNGMDELYK"
     CDS             7833..7889
                     /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             7890..8591
                     /codon_start=1
                     /product="monomeric blue fluorescent protein"
                     /label=TagBFP
                     /note="mammalian codon-optimized"
                     /translation="MSELIKENMHMKLYMEGTVDNHHFKCTSEGEGKPYEGTQTMRIKV
                     VEGGPLPFAFDILATSFLYGSKTFINHTQGIPDFFKQSFPEGFTWERVTTYEDGGVLTA
                     TQDTSLQDGYLIYNVKIRGVNFTSNGPVMQKKTLGWEAFTETLYPADGGLEGRNDMALK
                     LVGGSHLIANIKTTYRSKKPAKNLKMPGVYYVDYRLERIKEANNETYVEQHEVAVARYC
                     DLPSKLGHKLN"
     polyA_signal    8606..8830
                     /label=bGH poly(A) signal
                     /note="bovine growth hormone polyadenylation signal"
     primer_bind     9181..9200
                     /label=mPGK-F
                     /note="Mouse PGK promoter, forward primer"
     CDS             9256..9654
                     /codon_start=1
                     /gene="Aspergillus terreus BSD"
                     /product="blasticidin S deaminase"
                     /label=BSD
                     /note="confers resistance to blasticidin"
                     /translation="MAKPLSQEESTLIERATATINSIPISEDYSVASAALSSDGRIFTG
                     VNVYHFTGGPCAELVVLGTAAAAAAGNLTCIVAIGNENRGILSPCGRCRQVLLDLHPGI
                     KAIVKDSDGQPTAVGIRELLPSGYVWEG"
     misc_feature    9655..10243
                     /label=WPRE
                     /note="woodchuck hepatitis virus posttranscriptional 
                     regulatory element"
     primer_bind     complement(9708..9728)
                     /label=WPRE-R
                     /note="WPRE, reverse primer"
     CDS             complement(10126..10137)
                     /codon_start=1
                     /product="Factor Xa recognition and cleavage site"
                     /label=Factor Xa site
                     /translation="IEGR"
     repeat_region   10426..10488
                     /label=piggyBac right (3') inverted repeat
                     /note="piggyBac transposon-specific inverted terminal 
                     repeat sequence (ITR)"
     primer_bind     complement(10518..10538)
                     /label=T3
                     /note="T3 promoter, forward primer"
     promoter        complement(10518..10536)
                     /label=T3 promoter
                     /note="promoter for bacteriophage T3 RNA polymerase"
     primer_bind     complement(10557..10573)
                     /label=M13 rev
                     /note="common sequencing primer, one of multiple similar 
                     variants"
     primer_bind     complement(10557..10573)
                     /label=M13 Reverse
                     /note="In lacZ gene. Also called M13-rev"
     primer_bind     complement(10570..10592)
                     /label=M13/pUC Reverse
                     /note="In lacZ gene"
     protein_bind    10581..10597
                     /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(10605..10611,10612..10629,10630..10635))
                     /label=lac promoter
                     /note="promoter for the E. coli lac operon"
     protein_bind    10650..10671
                     /label=CAP binding site
                     /bound_moiety="E. coli catabolite activator protein"
                     /note="CAP binding activates transcription in the presence 
                     of cAMP."
     primer_bind     complement(10788..10805)
                     /label=L4440
                     /note="L4440 vector, forward primer"
     rep_origin      complement(10959..11547)
                     /direction=LEFT
                     /label=ori
                     /note="high-copy-number ColE1/pMB1/pBR322/pUC origin of 
                     replication"
     primer_bind     complement(11039..11058)
                     /label=pBR322ori-F
                     /note="pBR322 origin, forward primer"
     CDS             complement(join(11718..11929,1..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 ggccgcatca gtctgattgc ggcgttagcg gtagattacg
     2401 ttatcggcat ggaaaacgcc atgccgtgga acctgcctgc cgatctcgcc tggtttaaac
     2461 gcaacacctt aaataaaccc gtgattatgg gccgccatac ctgggaatca atcggtcgtc
     2521 cgttgccagg acgcaaaaat attatcctca gcagtcaacc gagtacggac gatcgcgtaa
     2581 cgtgggtgaa gtcggtggat gaagccatcg cggcgtgtgg tgacgtacca gaaatcatgg
     2641 tgattggcgg cggtcgcgtt attgaacagt tcttgccaaa agcgcaaaaa ctgtatctga
     2701 cgcatatcga cgcagaagtg gaaggcgaca cccatttccc ggattacgag ccggatgact
     2761 gggaatcggt attcagcgaa ttccacgatg ctgatgcgca gaactctcac agctattgct
     2821 ttgagattct ggagcggcga ggatccggcg gatctggagg tagtggtgga tctggaggtt
     2881 ctggtggctc cgacaagaag tacagcatcg gcctggccat cggcaccaac tctgtgggct
     2941 gggccgtgat caccgacgag tacaaggtgc ccagcaagaa attcaaggtg ctgggcaaca
     3001 ccgaccggca cagcatcaag aagaacctga tcggcgccct gctgttcgac agcggagaaa
     3061 cagccgaggc cacccggctg aagagaaccg ccagaagaag atacaccaga cggaagaacc
     3121 ggatctgcta tctgcaagag atcttcagca acgagatggc caaggtggac gacagcttct
     3181 tccacagact ggaagagtcc ttcctggtgg aagaggataa gaagcacgag cggcacccca
     3241 tcttcggcaa catcgtggac gaggtggcct accacgagaa gtaccccacc atctaccacc
     3301 tgagaaagaa actggtggac agcaccgaca aggccgacct gcggctgatc tatctggccc
     3361 tggcccacat gatcaagttc cggggccact tcctgatcga gggcgacctg aaccccgaca
     3421 acagcgacgt ggacaagctg ttcatccagc tggtgcagac ctacaaccag ctgttcgagg
     3481 aaaaccccat caacgccagc ggcgtggacg ccaaggccat cctgtctgcc agactgagca
     3541 agagcagacg gctggaaaat ctgatcgccc agctgcccgg cgagaagaag aatggcctgt
     3601 tcggcaacct gattgccctg agcctgggcc tgacccccaa cttcaagagc aacttcgacc
     3661 tggccgagga tgccaaactg cagctgagca aggacaccta cgacgacgac ctggacaacc
     3721 tgctggccca gatcggcgac cagtacgccg acctgtttct ggccgccaag aacctgtccg
     3781 acgccatcct gctgagcgac atcctgagag tgaacaccga gatcaccaag gcccccctga
     3841 gcgcctctat gatcaagaga tacgacgagc accaccagga cctgaccctg ctgaaagctc
     3901 tcgtgcggca gcagctgcct gagaagtaca aagagatttt cttcgaccag agcaagaacg
     3961 gctacgccgg ctacatcgat ggcggagcca gccaggaaga gttctacaag ttcatcaagc
     4021 ccatcctgga aaagatggac ggcaccgagg aactgctcgt gaagctgaac agagaggacc
     4081 tgctgcggaa gcagcggacc ttcgacaacg gcagcatccc ccaccagatc cacctgggag
     4141 agctgcacgc cattctgcgg cggcaggaag atttttaccc attcctgaag gacaaccggg
     4201 aaaagatcga gaagatcctg accttccgca tcccctacta cgtgggccct ctggccaggg
     4261 gaaacagcag attcgcctgg atgaccagaa agagcgagga aaccatcacc ccctggaact
     4321 tcgaggaagt ggtggacaag ggcgccagcg cccagagctt catcgagcgg atgaccaact
     4381 tcgataagaa cctgcccaac gagaaggtgc tgcccaagca cagcctgctg tacgagtact
     4441 tcaccgtgta caacgagctg accaaagtga aatacgtgac cgagggaatg agaaagcccg
     4501 ccttcctgag cggcgagcag aaaaaagcca tcgtggacct gctgttcaag accaaccgga
     4561 aagtgaccgt gaagcagctg aaagaggact acttcaagaa aatcgagtgc ttcgactccg
     4621 tggaaatctc cggcgtggaa gatcggttca acgcctccct gggcacatac cacgatctgc
     4681 tgaaaattat caaggacaag gacttcctgg acaatgagga aaacgaggac attctggaag
     4741 atatcgtgct gaccctgaca ctgtttgagg acagagagat gatcgaggaa cggctgaaaa
     4801 cctatgccca cctgttcgac gacaaagtga tgaagcagct gaagcggcgg agatacaccg
     4861 gctggggcag gctgagccgg aagctgatca acggcatccg ggacaagcag tccggcaaga
     4921 caatcctgga tttcctgaag tccgacggct tcgccaacag aaacttcatg cagctgatcc
     4981 acgacgacag cctgaccttt aaagaggaca tccagaaagc ccaggtgtcc ggccagggcg
     5041 atagcctgca cgagcacatt gccaatctgg ccggcagccc cgccattaag aagggcatcc
     5101 tgcagacagt gaaggtggtg gacgagctcg tgaaagtgat gggccggcac aagcccgaga
     5161 acatcgtgat cgaaatggcc agagagaacc agaccaccca gaagggacag aagaacagcc
     5221 gcgagagaat gaagcggatc gaagagggca tcaaagagct gggcagccag atcctgaaag
     5281 aacaccccgt ggaaaacacc cagctgcaga acgagaagct gtacctgtac tacctgcaga
     5341 atgggcggga tatgtacgtg gaccaggaac tggacatcaa ccggctgtcc gactacgatg
     5401 tggacgctat cgtgcctcag agctttctga aggacgactc catcgataac aaagtgctga
     5461 ctcggagcga caagaaccgg ggcaagagcg acaacgtgcc ctccgaagag gtcgtgaaga
     5521 agatgaagaa ctactggcgc cagctgctga atgccaagct gattacccag aggaagttcg
     5581 acaatctgac caaggccgag agaggcggcc tgagcgaact ggataaggcc ggcttcatca
     5641 agagacagct ggtggaaacc cggcagatca caaagcacgt ggcacagatc ctggactccc
     5701 ggatgaacac taagtacgac gagaacgaca aactgatccg ggaagtgaaa gtgatcaccc
     5761 tgaagtccaa gctggtgtcc gatttccgga aggatttcca gttttacaaa gtgcgcgaga
     5821 tcaacaacta ccaccacgcc cacgacgcct acctgaacgc cgtcgtggga accgccctga
     5881 tcaaaaagta ccctaagctg gaaagcgagt tcgtgtacgg cgactacaag gtgtacgacg
     5941 tgcggaagat gatcgccaag agcgagcagg aaatcggcaa ggctaccgcc aagtacttct
     6001 tctacagcaa catcatgaac tttttcaaga ccgagattac cctggccaac ggcgagatcc
     6061 ggaagcggcc tctgatcgag acaaacggcg aaacaggcga gatcgtgtgg gataagggcc
     6121 gggactttgc caccgtgcgg aaagtgctgt ctatgcccca agtgaatatc gtgaaaaaga
     6181 ccgaggtgca gacaggcggc ttcagcaaag agtctatcct gcccaagagg aacagcgaca
     6241 agctgatcgc cagaaagaag gactgggacc ctaagaagta cggcggcttc gacagcccca
     6301 ccgtggccta ttctgtgctg gtggtggcca aagtggaaaa gggcaagtcc aagaaactga
     6361 agagtgtgaa agagctgctg gggatcacca tcatggaaag aagcagcttc gagaagaatc
     6421 ccatcgactt tctggaagcc aagggctaca aagaagtgaa aaaggacctg atcatcaagc
     6481 tgcctaagta ctccctgttc gagctggaaa acggccggaa gagaatgctg gcctctgccg
     6541 gcgaactgca gaagggaaac gaactggccc tgccctccaa atatgtgaac ttcctgtacc
     6601 tggccagcca ctatgagaag ctgaagggct cccccgagga taatgagcag aaacagctgt
     6661 ttgtggaaca gcacaaacac tacctggacg agatcatcga gcagatcagc gagttctcca
     6721 agagagtgat cctggccgac gctaatctgg acaaggtgct gagcgcctac aacaagcaca
     6781 gagacaagcc tatcagagag caggccgaga atatcatcca cctgtttacc ctgaccaatc
     6841 tgggagcccc tgccgccttc aagtactttg acaccaccat cgaccggaag aggtacacca
     6901 gcaccaaaga ggtgctggac gccaccctga tccaccagag catcaccggc ctgtacgaga
     6961 cacggatcga cctgtctcag ctgggaggcg accccaagaa gaagcgcaag gtgcaccacc
     7021 accaccacca cggaggaggt ggaagcggag gaggaggaag cggaggagga ggtagctggc
     7081 gcgccgtgtc taagggcgaa gagctgatta aggagaacat gcacatgaag ctgtacatgg
     7141 agggcaccgt gaacaaccac cacttcaagt gcacatccga gggcgaaggc aagccctacg
     7201 agggcaccca gaccatgaga atcaaggtgg tcgagggcgg ccctctcccc ttcgccttcg
     7261 acatcctggc taccagcttc atgtacggca gcagaacctt catcaaccac acccagggca
     7321 tccccgactt ctttaagcag tccttccctg agggcttcac atgggagaga gtcaccacat
     7381 acgaagacgg gggcgtgctg accgctaccc aggacaccag cctccaggac ggctgcctca
     7441 tctacaacgt caagatcaga ggggtgaact tcccatccaa cggccctgtg atgcagaaga
     7501 aaacactcgg ctgggaggcc aacaccgaga tgctgtaccc cgctgacggc ggcctggaag
     7561 gcagaaccga catggccctg aagctcgtgg gcgggggcca cctgatctgc aacttcaaga
     7621 ccacatacag atccaagaaa cccgctaaga acctcaagat gcccggcgtc tactatgtgg
     7681 accacagact ggaaagaatc aaggaggccg acaaagagac ctacgtcgag cagcacgagg
     7741 tggctgtggc cagatactgc gacctcccta gcaaactggg gcacaaactt aatggcatgg
     7801 acgagctgta caagttaatt aacggaagcg gagctactaa cttcagcctg ctgaagcagg
     7861 ctggagacgt ggaggagaac cctggaccta tgagcgagct gattaaggag aacatgcaca
     7921 tgaagctgta catggagggc accgtggaca accatcactt caagtgcaca tccgagggcg
     7981 aaggcaagcc ctacgagggc acccagacca tgagaatcaa ggtggtcgag ggcggccctc
     8041 tccccttcgc cttcgacatc ctggctacta gcttcctcta cggcagcaag accttcatca
     8101 accacaccca gggcatcccc gacttcttca agcagtcctt ccctgagggc ttcacatggg
     8161 agagagtcac cacatacgaa gacgggggcg tgctgaccgc tacccaggac accagcctcc
     8221 aggacggcta cctcatctac aacgtcaaga tcagaggggt gaacttcaca tccaacggcc
     8281 ctgtgatgca gaagaaaaca ctcggctggg aggccttcac cgagacgctg taccccgctg
     8341 acggcggcct ggaaggcaga aacgacatgg ccctgaagct cgtgggcggg agccatctga
     8401 tcgcaaacat caagaccaca tatagatcca agaaacccgc taagaacctc aagatgcctg
     8461 gcgtctacta tgtggactac agactggaaa gaatcaagga ggccaacaac gagacctacg
     8521 tcgagcagca cgaggtggca gtggccagat actgcgacct ccctagcaaa ctggggcaca
     8581 agcttaatta gacgcgtatt aacgactgtg ccttctagtt gccagccatc tgttgtttgc
     8641 ccctcccccg tgccttcctt gaccctggaa ggtgccactc ccactgtcct ttcctaataa
     8701 aatgaggaaa ttgcatcgca ttgtctgagt aggtgtcatt ctattctggg gggtggggtg
     8761 gggcaggaca gcaaggggga ggattgggaa gacaatagca ggcatgctgg ggatgcggtg
     8821 ggctctatgg cctctggcct cgcacacatt ccacatccac cggtaggcgc caaccggctc
     8881 cgttctttgg tggccccttc gcgccacctt ctactcctcc cctagtcagg aagttccccc
     8941 ccgccccgca gctcgcgtcg tgcaggacgt gacaaatgga agtagcacgt ctcactagtc
     9001 tcgtgcagat ggacagcacc gctgagcaat ggaagcgggt aggcctttgg ggcagcggcc
     9061 aatagcagct ttgctccttc gctttctggg ctcagaggct gggaaggggt gggtccgggg
     9121 gcgggctcag gggcgggctc aggggcgggg cgggcgcccg aaggtcctcc ggaggcccgg
     9181 cattctgcac gcttcaaaag cgcacgtctg ccgcgctgtt ctcctcttcc tcatctccgg
     9241 gcctttcgac ctgcaatggc caagcctttg tctcaagaag aatccaccct cattgaaaga
     9301 gcaacggcta caatcaacag catccccatc tctgaagact acagcgtcgc cagcgcagct
     9361 ctctctagcg acggccgcat cttcactggt gtcaatgtat atcattttac tgggggacct
     9421 tgtgcagaac tcgtggtgct gggcactgct gctgctgcgg cagctggcaa cctgacttgt
     9481 atcgtcgcga tcggaaatga gaacaggggc atcttgagcc cctgcggacg gtgccgacag
     9541 gtgcttctcg atctgcatcc tgggatcaaa gccatagtga aggacagtga tggacagccg
     9601 acggcagttg ggattcgtga attgctgccc tctggttatg tgtgggaggg ctaaaatcaa
     9661 cctctggatt acaaaatttg tgaaagattg actggtattc ttaactatgt tgctcctttt
     9721 acgctatgtg gatacgctgc tttaatgcct ttgtatcatg ctattgcttc ccgtatggct
     9781 ttcattttct cctccttgta taaatcctgg ttgctgtctc tttatgagga gttgtggccc
     9841 gttgtcaggc aacgtggcgt ggtgtgcact gtgtttgctg acgcaacccc cactggttgg
     9901 ggcattgcca ccacctgtca gctcctttcc gggactttcg ctttccccct ccctattgcc
     9961 acggcggaac tcatcgccgc ctgccttgcc cgctgctgga caggggctcg gctgttgggc
    10021 actgacaatt ccgtggtgtt gtcggggaaa tcatcgtcct ttccttggct gctcgcctgt
    10081 gttgccacct ggattctgcg cgggacgtcc ttctgctacg tcccttcggc cctcaatcca
    10141 gcggaccttc cttcccgcgg cctgctgccg gctctgcggc ctcttccgcg tctccgcctt
    10201 cgccctcaga cgagtcggat ctccctttgg gccgcctccc cgccggtacc taaaagtttt
    10261 gttactttat agaagaaatt ttgagttttt gttttttttt aataaataaa taaacataaa
    10321 taaattgttt gttgaattta ttattagtat gtaagtgtaa atataataaa acttaatatc
    10381 tattcaaatt aataaataaa cctcgatata cagaccgata aaacacatgc gtcaatttta
    10441 cgcatgatta tctttaacgt acgtcacaat atgattatct ttctagggtt aatctagtat
    10501 taatccagct tttgttccct ttagtgaggg ttaattgcgc gcttggcgta atcatggtca
    10561 tagctgtttc ctgtgtgaaa ttgttatccg ctcacaattc cacacaacat acgagccgga
    10621 agcataaagt gtaaagcctg gggtgcctaa tgagtgagct aactcacatt aattgcgttg
    10681 cgctcactgc ccgctttcca gtcgggaaac ctgtcgtgcc agctgcatta atgaatcggc
    10741 caacgcgcgg ggagaggcgg tttgcgtatt gggcgctctt ccgcttcctc gctcactgac
    10801 tcgctgcgct cggtcgttcg gctgcggcga gcggtatcag ctcactcaaa ggcggtaata
    10861 cggttatcca cagaatcagg ggataacgca ggaaagaaca tgtgagcaaa aggccagcaa
    10921 aaggccagga accgtaaaaa ggccgcgttg ctggcgtttt tccataggct ccgcccccct
    10981 gacgagcatc acaaaaatcg acgctcaagt cagaggtggc gaaacccgac aggactataa
    11041 agataccagg cgtttccccc tggaagctcc ctcgtgcgct ctcctgttcc gaccctgccg
    11101 cttaccggat acctgtccgc ctttctccct tcgggaagcg tggcgctttc tcatagctca
    11161 cgctgtaggt atctcagttc ggtgtaggtc gttcgctcca agctgggctg tgtgcacgaa
    11221 ccccccgttc agcccgaccg ctgcgcctta tccggtaact atcgtcttga gtccaacccg
    11281 gtaagacacg acttatcgcc actggcagca gccactggta acaggattag cagagcgagg
    11341 tatgtaggcg gtgctacaga gttcttgaag tggtggccta actacggcta cactagaaga
    11401 acagtatttg gtatctgcgc tctgctgaag ccagttacct tcggaaaaag agttggtagc
    11461 tcttgatccg gcaaacaaac caccgctggt agcggtggtt tttttgtttg caagcagcag
    11521 attacgcgca gaaaaaaagg atctcaagaa gatcctttga tcttttctac ggggtctgac
    11581 gctcagtgga acgaaaactc acgttaaggg attttggtca tgagattatc aaaaaggatc
    11641 ttcacctaga tccttttaaa ttaaaaatga agttttaaat caatctaaag tatatatgag
    11701 taaacttggt ctgacagtta ccaatgctta atcagtgagg cacctatctc agcgatctgt
    11761 ctatttcgtt catccatagt tgcctgactc cccgtcgtgt agataactac gatacgggag
    11821 ggcttaccat ctggccccag tgctgcaatg ataccgcgag acccacgctc accggctcca
    11881 gatttatcag caataaacca gccagccgga agggccgagc gcagaagtg
//
