LOCUS       sequence-373500-       12343 bp    DNA     circular SYN 15-DEC-2025
DEFINITION  SMASh 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 12343)
  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 12343)
  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-SMASh-SpdCas9-tagRFPt-P2A-tagBFP-PGK-Blasticidin
FEATURES             Location/Qualifiers
     source          1..12343
                     /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..2393
                     /codon_start=1
                     /product="HA (human influenza hemagglutinin) epitope tag"
                     /label=HA
                     /translation="YPYDVPDYA"
     CDS             join(3318..3341,3342..3344,3345..5831,5832..5834,
                     5835..7418)
                     /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             3318..7418
                     /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             7419..7439
                     /codon_start=1
                     /product="nuclear localization signal of SV40 (simian virus
                     40) large T antigen"
                     /label=SV40 NLS
                     /translation="PKKKRKV"
     CDS             7440..7457
                     /codon_start=1
                     /product="6xHis affinity tag"
                     /label=6xHis
                     /translation="HHHHHH"
     CDS             7512..8240
                     /codon_start=1
                     /product="photostable variant of TagRFP (Shaner et al., 
                     2008)"
                     /label=TagRFP-T
                     /translation="VSKGEELIKENMHMKLYMEGTVNNHHFKCTSEGEGKPYEGTQTMR
                     IKVVEGGPLPFAFDILATSFMYGSRTFINHTQGIPDFFKQSFPEGFTWERVTTYEDGGV
                     LTATQDTSLQDGCLIYNVKIRGVNFPSNGPVMQKKTLGWEANTEMLYPADGGLEGRTDM
                     ALKLVGGGHLICNFKTTYRSKKPAKNLKMPGVYYVDHRLERIKEADKETYVEQHEVAVA
                     RYCDLPSKLGHKLNGMDELYK"
     CDS             8259..8315
                     /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             8316..9017
                     /codon_start=1
                     /product="monomeric blue fluorescent protein"
                     /label=TagBFP
                     /note="mammalian codon-optimized"
                     /translation="MSELIKENMHMKLYMEGTVDNHHFKCTSEGEGKPYEGTQTMRIKV
                     VEGGPLPFAFDILATSFLYGSKTFINHTQGIPDFFKQSFPEGFTWERVTTYEDGGVLTA
                     TQDTSLQDGCLIYNVKIRGVNFTSNGPVMQKKTLGWEAFTETLYPADGGLEGRNDMALK
                     LVGGSHLIANIKTTYRSKKPAKNLKMPGVYYVDYRLERIKEANNETYVEQHEVAVARYC
                     DLPSKLGHKLN"
     polyA_signal    9032..9256
                     /label=bGH poly(A) signal
                     /note="bovine growth hormone polyadenylation signal"
     primer_bind     9595..9614
                     /label=mPGK-F
                     /note="Mouse PGK promoter, forward primer"
     CDS             9670..10068
                     /codon_start=1
                     /gene="Aspergillus terreus BSD"
                     /product="blasticidin S deaminase"
                     /label=BSD
                     /note="confers resistance to blasticidin"
                     /translation="MAKPLSQEESTLIERATATINSIPISEDYSVASAALSSDGRIFTG
                     VNVYHFTGGPCAELVVLGTAAAAAAGNLTCIVAIGNENRGILSPCGRCRQVLLDLHPGI
                     KAIVKDSDGQPTAVGIRELLPSGYVWEG"
     misc_feature    10069..10657
                     /label=WPRE
                     /note="woodchuck hepatitis virus posttranscriptional 
                     regulatory element"
     primer_bind     complement(10122..10142)
                     /label=WPRE-R
                     /note="WPRE, reverse primer"
     CDS             complement(10540..10551)
                     /codon_start=1
                     /product="Factor Xa recognition and cleavage site"
                     /label=Factor Xa site
                     /translation="IEGR"
     repeat_region   10840..10902
                     /label=piggyBac right (3') inverted repeat
                     /note="piggyBac transposon-specific inverted terminal 
                     repeat sequence (ITR)"
     primer_bind     complement(10932..10952)
                     /label=T3
                     /note="T3 promoter, forward primer"
     promoter        complement(10932..10950)
                     /label=T3 promoter
                     /note="promoter for bacteriophage T3 RNA polymerase"
     primer_bind     complement(10971..10987)
                     /label=M13 rev
                     /note="common sequencing primer, one of multiple similar 
                     variants"
     primer_bind     complement(10971..10987)
                     /label=M13 Reverse
                     /note="In lacZ gene. Also called M13-rev"
     primer_bind     complement(10984..11006)
                     /label=M13/pUC Reverse
                     /note="In lacZ gene"
     protein_bind    10995..11011
                     /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(11019..11025,11026..11043,11044..11049))
                     /label=lac promoter
                     /note="promoter for the E. coli lac operon"
     protein_bind    11064..11085
                     /label=CAP binding site
                     /bound_moiety="E. coli catabolite activator protein"
                     /note="CAP binding activates transcription in the presence 
                     of cAMP."
     primer_bind     complement(11202..11219)
                     /label=L4440
                     /note="L4440 vector, forward primer"
     rep_origin      complement(11373..11961)
                     /direction=LEFT
                     /label=ori
                     /note="high-copy-number ColE1/pMB1/pBR322/pUC origin of 
                     replication"
     primer_bind     complement(11453..11472)
                     /label=pBR322ori-F
                     /note="pBR322 origin, forward primer"
     CDS             complement(join(12132..12343,1..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 ggccgctacc catacgatgt tccagattac gctggaggta
     2401 gtggcggaag tggtgggagc gcgcccatca cggcgtacgc ccagcagacg agaggcctcc
     2461 tagggtgtat aatcaccagc ctgactggcc gggacaaaaa ccaagtggag ggtgaggtcc
     2521 agatcgtgtc aactgctacc caaaccttcc tggcaacgtg catcaatggg gtatgctggg
     2581 cagtctacca cggggccgga acgaggacca tcgcatcacc caagggtcct gtcatccaga
     2641 tgtataccaa tgtggaccaa gaccttgtgg gctggcccgc tcctcaaggt tcccgctcat
     2701 tgacaccctg tacctgcggc tcctcggacc tttacctggt cacgaggcac gccgatgtca
     2761 ttcccgtgcg ccggcgaggt gatagcaggg gtagcctgct ttcgccccgg cccatttcct
     2821 acttgaaagg ctcctcgggg ggtccgctgt tgtgccccgc gggacacgcc gtgggcctat
     2881 tcagggccgc ggtgtgcacc cgtggagtgg ctaaagcggt ggactttatc cctgtggaga
     2941 acctagagac aaccatgaga tccccggtgt tcacggacaa ctcctctcca ccagcagtca
     3001 ccctgacgca cccaatcacc aaaatcgata ccaaatacat catgacatgc atgtcggccg
     3061 acctggaggt cgtcacgagc acctgggtgc tcgttggcgg cgtcctggct gctctggccg
     3121 cgtattgcct gtcaacaggc tgcgtggtca tagtgggcag gatcgtcttg tccgggaagc
     3181 cggcaggcag tagcggaagc agtattatac ctgacaggga ggttctctac caggagttcg
     3241 aagatgtcgt gccatgctca atgggcggat ccggcggatc tggaggtagt ggtggatctg
     3301 gaggttctgg tggctccgac aagaagtaca gcatcggcct ggccatcggc accaactctg
     3361 tgggctgggc cgtgatcacc gacgagtaca aggtgcccag caagaaattc aaggtgctgg
     3421 gcaacaccga ccggcacagc atcaagaaga acctgatcgg cgccctgctg ttcgacagcg
     3481 gagaaacagc cgaggccacc cggctgaaga gaaccgccag aagaagatac accagacgga
     3541 agaaccggat ctgctatctg caagagatct tcagcaacga gatggccaag gtggacgaca
     3601 gcttcttcca cagactggaa gagtccttcc tggtggaaga ggataagaag cacgagcggc
     3661 accccatctt cggcaacatc gtggacgagg tggcctacca cgagaagtac cccaccatct
     3721 accacctgag aaagaaactg gtggacagca ccgacaaggc cgacctgcgg ctgatctatc
     3781 tggccctggc ccacatgatc aagttccggg gccacttcct gatcgagggc gacctgaacc
     3841 ccgacaacag cgacgtggac aagctgttca tccagctggt gcagacctac aaccagctgt
     3901 tcgaggaaaa ccccatcaac gccagcggcg tggacgccaa ggccatcctg tctgccagac
     3961 tgagcaagag cagacggctg gaaaatctga tcgcccagct gcccggcgag aagaagaatg
     4021 gcctgttcgg caacctgatt gccctgagcc tgggcctgac ccccaacttc aagagcaact
     4081 tcgacctggc cgaggatgcc aaactgcagc tgagcaagga cacctacgac gacgacctgg
     4141 acaacctgct ggcccagatc ggcgaccagt acgccgacct gtttctggcc gccaagaacc
     4201 tgtccgacgc catcctgctg agcgacatcc tgagagtgaa caccgagatc accaaggccc
     4261 ccctgagcgc ctctatgatc aagagatacg acgagcacca ccaggacctg accctgctga
     4321 aagctctcgt gcggcagcag ctgcctgaga agtacaaaga gattttcttc gaccagagca
     4381 agaacggcta cgccggctac atcgatggcg gagccagcca ggaagagttc tacaagttca
     4441 tcaagcccat cctggaaaag atggacggca ccgaggaact gctcgtgaag ctgaacagag
     4501 aggacctgct gcggaagcag cggaccttcg acaacggcag catcccccac cagatccacc
     4561 tgggagagct gcacgccatt ctgcggcggc aggaagattt ttacccattc ctgaaggaca
     4621 accgggaaaa gatcgagaag atcctgacct tccgcatccc ctactacgtg ggccctctgg
     4681 ccaggggaaa cagcagattc gcctggatga ccagaaagag cgaggaaacc atcaccccct
     4741 ggaacttcga ggaagtggtg gacaagggcg ccagcgccca gagcttcatc gagcggatga
     4801 ccaacttcga taagaacctg cccaacgaga aggtgctgcc caagcacagc ctgctgtacg
     4861 agtacttcac cgtgtacaac gagctgacca aagtgaaata cgtgaccgag ggaatgagaa
     4921 agcccgcctt cctgagcggc gagcagaaaa aagccatcgt ggacctgctg ttcaagacca
     4981 accggaaagt gaccgtgaag cagctgaaag aggactactt caagaaaatc gagtgcttcg
     5041 actccgtgga aatctccggc gtggaagatc ggttcaacgc ctccctgggc acataccacg
     5101 atctgctgaa aattatcaag gacaaggact tcctggacaa tgaggaaaac gaggacattc
     5161 tggaagatat cgtgctgacc ctgacactgt ttgaggacag agagatgatc gaggaacggc
     5221 tgaaaaccta tgcccacctg ttcgacgaca aagtgatgaa gcagctgaag cggcggagat
     5281 acaccggctg gggcaggctg agccggaagc tgatcaacgg catccgggac aagcagtccg
     5341 gcaagacaat cctggatttc ctgaagtccg acggcttcgc caacagaaac ttcatgcagc
     5401 tgatccacga cgacagcctg acctttaaag aggacatcca gaaagcccag gtgtccggcc
     5461 agggcgatag cctgcacgag cacattgcca atctggccgg cagccccgcc attaagaagg
     5521 gcatcctgca gacagtgaag gtggtggacg agctcgtgaa agtgatgggc cggcacaagc
     5581 ccgagaacat cgtgatcgaa atggccagag agaaccagac cacccagaag ggacagaaga
     5641 acagccgcga gagaatgaag cggatcgaag agggcatcaa agagctgggc agccagatcc
     5701 tgaaagaaca ccccgtggaa aacacccagc tgcagaacga gaagctgtac ctgtactacc
     5761 tgcagaatgg gcgggatatg tacgtggacc aggaactgga catcaaccgg ctgtccgact
     5821 acgatgtgga cgctatcgtg cctcagagct ttctgaagga cgactccatc gataacaaag
     5881 tgctgactcg gagcgacaag aaccggggca agagcgacaa cgtgccctcc gaagaggtcg
     5941 tgaagaagat gaagaactac tggcgccagc tgctgaatgc caagctgatt acccagagga
     6001 agttcgacaa tctgaccaag gccgagagag gcggcctgag cgaactggat aaggccggct
     6061 tcatcaagag acagctggtg gaaacccggc agatcacaaa gcacgtggca cagatcctgg
     6121 actcccggat gaacactaag tacgacgaga acgacaaact gatccgggaa gtgaaagtga
     6181 tcaccctgaa gtccaagctg gtgtccgatt tccggaagga tttccagttt tacaaagtgc
     6241 gcgagatcaa caactaccac cacgcccacg acgcctacct gaacgccgtc gtgggaaccg
     6301 ccctgatcaa aaagtaccct aagctggaaa gcgagttcgt gtacggcgac tacaaggtgt
     6361 acgacgtgcg gaagatgatc gccaagagcg agcaggaaat cggcaaggct accgccaagt
     6421 acttcttcta cagcaacatc atgaactttt tcaagaccga gattaccctg gccaacggcg
     6481 agatccggaa gcggcctctg atcgagacaa acggcgaaac aggcgagatc gtgtgggata
     6541 agggccggga ctttgccacc gtgcggaaag tgctgtctat gccccaagtg aatatcgtga
     6601 aaaagaccga ggtgcagaca ggcggcttca gcaaagagtc tatcctgccc aagaggaaca
     6661 gcgacaagct gatcgccaga aagaaggact gggaccctaa gaagtacggc ggcttcgaca
     6721 gccccaccgt ggcctattct gtgctggtgg tggccaaagt ggaaaagggc aagtccaaga
     6781 aactgaagag tgtgaaagag ctgctgggga tcaccatcat ggaaagaagc agcttcgaga
     6841 agaatcccat cgactttctg gaagccaagg gctacaaaga agtgaaaaag gacctgatca
     6901 tcaagctgcc taagtactcc ctgttcgagc tggaaaacgg ccggaagaga atgctggcct
     6961 ctgccggcga actgcagaag ggaaacgaac tggccctgcc ctccaaatat gtgaacttcc
     7021 tgtacctggc cagccactat gagaagctga agggctcccc cgaggataat gagcagaaac
     7081 agctgtttgt ggaacagcac aaacactacc tggacgagat catcgagcag atcagcgagt
     7141 tctccaagag agtgatcctg gccgacgcta atctggacaa ggtgctgagc gcctacaaca
     7201 agcacagaga caagcctatc agagagcagg ccgagaatat catccacctg tttaccctga
     7261 ccaatctggg agcccctgcc gccttcaagt actttgacac caccatcgac cggaagaggt
     7321 acaccagcac caaagaggtg ctggacgcca ccctgatcca ccagagcatc accggcctgt
     7381 acgagacacg gatcgacctg tctcagctgg gaggcgaccc caagaagaag cgcaaggtgc
     7441 accaccacca ccaccacgga ggaggtggaa gcggaggagg aggaagcgga ggaggaggta
     7501 gctggcgcgc cgtgtctaag ggcgaagagc tgattaagga gaacatgcac atgaagctgt
     7561 acatggaggg caccgtgaac aaccaccact tcaagtgcac atccgagggc gaaggcaagc
     7621 cctacgaggg cacccagacc atgagaatca aggtggtcga gggcggccct ctccccttcg
     7681 ccttcgacat cctggctacc agcttcatgt acggcagcag aaccttcatc aaccacaccc
     7741 agggcatccc cgacttcttt aagcagtcct tccctgaggg cttcacatgg gagagagtca
     7801 ccacatacga agacgggggc gtgctgaccg ctacccagga caccagcctc caggacggct
     7861 gcctcatcta caacgtcaag atcagagggg tgaacttccc atccaacggc cctgtgatgc
     7921 agaagaaaac actcggctgg gaggccaaca ccgagatgct gtaccccgct gacggcggcc
     7981 tggaaggcag aaccgacatg gccctgaagc tcgtgggcgg gggccacctg atctgcaact
     8041 tcaagaccac atacagatcc aagaaacccg ctaagaacct caagatgccc ggcgtctact
     8101 atgtggacca cagactggaa agaatcaagg aggccgacaa agagacctac gtcgagcagc
     8161 acgaggtggc tgtggccaga tactgcgacc tccctagcaa actggggcac aaacttaatg
     8221 gcatggacga gctgtacaag ttaattaacg gaagcggagc tactaacttc agcctgctga
     8281 agcaggctgg agacgtggag gagaaccctg gacctatgag cgagctgatt aaggagaaca
     8341 tgcacatgaa gctgtacatg gagggcaccg tggacaacca tcacttcaag tgcacatccg
     8401 agggcgaagg caagccctac gagggcaccc agaccatgag aatcaaggtg gtcgagggcg
     8461 gccctctccc cttcgccttc gacatcctgg ctactagctt cctctacggc agcaagacct
     8521 tcatcaacca cacccagggc atccccgact tcttcaagca gtccttccct gagggcttca
     8581 catgggagag agtcaccaca tacgaagacg ggggcgtgct gaccgctacc caggacacca
     8641 gcctccagga cggctgcctc atctacaacg tcaagatcag aggggtgaac ttcacatcca
     8701 acggccctgt gatgcagaag aaaacactcg gctgggaggc cttcaccgag acgctgtacc
     8761 ccgctgacgg cggcctggaa ggcagaaacg acatggccct gaagctcgtg ggcgggagcc
     8821 atctgatcgc aaacatcaag accacatata gatccaagaa acccgctaag aacctcaaga
     8881 tgcctggcgt ctactatgtg gactacagac tggaaagaat caaggaggcc aacaacgaga
     8941 cctacgtcga gcagcacgag gtggcagtgg ccagatactg cgacctccct agcaaactgg
     9001 ggcacaagct taattagacg cgtattaacg actgtgcctt ctagttgcca gccatctgtt
     9061 gtttgcccct cccccgtgcc ttccttgacc ctggaaggtg ccactcccac tgtcctttcc
     9121 taataaaatg aggaaattgc atcgcattgt ctgagtaggt gtcattctat tctggggggt
     9181 ggggtggggc aggacagcaa gggggaggat tgggaagaca atagcaggca tgctggggat
     9241 gcggtgggct ctatggcctc tggcctcgca cacattccac atccaccggt aggcgccaac
     9301 cggctccgtt ctttggtggc cccttcgcgc caccttctac tcctccccta gtcaggaagt
     9361 tcccccccgc cccgcagctc gcgtcgtgca ggacgtgaca aatggaagta gcacgtctca
     9421 ctagtctcgt gcagatggac agcaccgctg agcaatggaa gcgggtaggc ctttggggca
     9481 gcggccaata gcagctttgc tccttcgctt tctgggctca gaggctggga aggggtgggt
     9541 ccgggggcgg gctcaggggc ggggcgggcg cccgaaggtc ctccggaggc ccggcattct
     9601 gcacgcttca aaagcgcacg tctgccgcgc tgttctcctc ttcctcatct ccgggccttt
     9661 cgacctgcaa tggccaagcc tttgtctcaa gaagaatcca ccctcattga aagagcaacg
     9721 gctacaatca acagcatccc catctctgaa gactacagcg tcgccagcgc agctctctct
     9781 agcgacggcc gcatcttcac tggtgtcaat gtatatcatt ttactggggg accttgtgca
     9841 gaactcgtgg tgctgggcac tgctgctgct gcggcagctg gcaacctgac ttgtatcgtc
     9901 gcgatcggaa atgagaacag gggcatcttg agcccctgcg gacggtgccg acaggtgctt
     9961 ctcgatctgc atcctgggat caaagccata gtgaaggaca gtgatggaca gccgacggca
    10021 gttgggattc gtgaattgct gccctctggt tatgtgtggg agggctaaaa tcaacctctg
    10081 gattacaaaa tttgtgaaag attgactggt attcttaact atgttgctcc ttttacgcta
    10141 tgtggatacg ctgctttaat gcctttgtat catgctattg cttcccgtat ggctttcatt
    10201 ttctcctcct tgtataaatc ctggttgctg tctctttatg aggagttgtg gcccgttgtc
    10261 aggcaacgtg gcgtggtgtg cactgtgttt gctgacgcaa cccccactgg ttggggcatt
    10321 gccaccacct gtcagctcct ttccgggact ttcgctttcc ccctccctat tgccacggcg
    10381 gaactcatcg ccgcctgcct tgcccgctgc tggacagggg ctcggctgtt gggcactgac
    10441 aattccgtgg tgttgtcggg gaaatcatcg tcctttcctt ggctgctcgc ctgtgttgcc
    10501 acctggattc tgcgcgggac gtccttctgc tacgtccctt cggccctcaa tccagcggac
    10561 cttccttccc gcggcctgct gccggctctg cggcctcttc cgcgtctccg ccttcgccct
    10621 cagacgagtc ggatctccct ttgggccgcc tccccgccgg tacctaaaag ttttgttact
    10681 ttatagaaga aattttgagt ttttgttttt ttttaataaa taaataaaca taaataaatt
    10741 gtttgttgaa tttattatta gtatgtaagt gtaaatataa taaaacttaa tatctattca
    10801 aattaataaa taaacctcga tatacagacc gataaaacac atgcgtcaat tttacgcatg
    10861 attatcttta acgtacgtca caatatgatt atctttctag ggttaatcta gtattaatcc
    10921 agcttttgtt ccctttagtg agggttaatt gcgcgcttgg cgtaatcatg gtcatagctg
    10981 tttcctgtgt gaaattgtta tccgctcaca attccacaca acatacgagc cggaagcata
    11041 aagtgtaaag cctggggtgc ctaatgagtg agctaactca cattaattgc gttgcgctca
    11101 ctgcccgctt tccagtcggg aaacctgtcg tgccagctgc attaatgaat cggccaacgc
    11161 gcggggagag gcggtttgcg tattgggcgc tcttccgctt cctcgctcac tgactcgctg
    11221 cgctcggtcg ttcggctgcg gcgagcggta tcagctcact caaaggcggt aatacggtta
    11281 tccacagaat caggggataa cgcaggaaag aacatgtgag caaaaggcca gcaaaaggcc
    11341 aggaaccgta aaaaggccgc gttgctggcg tttttccata ggctccgccc ccctgacgag
    11401 catcacaaaa atcgacgctc aagtcagagg tggcgaaacc cgacaggact ataaagatac
    11461 caggcgtttc cccctggaag ctccctcgtg cgctctcctg ttccgaccct gccgcttacc
    11521 ggatacctgt ccgcctttct cccttcggga agcgtggcgc tttctcatag ctcacgctgt
    11581 aggtatctca gttcggtgta ggtcgttcgc tccaagctgg gctgtgtgca cgaacccccc
    11641 gttcagcccg accgctgcgc cttatccggt aactatcgtc ttgagtccaa cccggtaaga
    11701 cacgacttat cgccactggc agcagccact ggtaacagga ttagcagagc gaggtatgta
    11761 ggcggtgcta cagagttctt gaagtggtgg cctaactacg gctacactag aagaacagta
    11821 tttggtatct gcgctctgct gaagccagtt accttcggaa aaagagttgg tagctcttga
    11881 tccggcaaac aaaccaccgc tggtagcggt ggtttttttg tttgcaagca gcagattacg
    11941 cgcagaaaaa aaggatctca agaagatcct ttgatctttt ctacggggtc tgacgctcag
    12001 tggaacgaaa actcacgtta agggattttg gtcatgagat tatcaaaaag gatcttcacc
    12061 tagatccttt taaattaaaa atgaagtttt aaatcaatct aaagtatata tgagtaaact
    12121 tggtctgaca gttaccaatg cttaatcagt gaggcaccta tctcagcgat ctgtctattt
    12181 cgttcatcca tagttgcctg actccccgtc gtgtagataa ctacgatacg ggagggctta
    12241 ccatctggcc ccagtgctgc aatgataccg cgagacccac gctcaccggc tccagattta
    12301 tcagcaataa accagccagc cggaagggcc gagcgcagaa gtg
//
