LOCUS       sequence-304742-       11241 bp    DNA     circular SYN 15-DEC-2025
DEFINITION  Tunable and temporal expression control of dCas9.
ACCESSION   .
VERSION     .
KEYWORDS    .
SOURCE      synthetic DNA construct
  ORGANISM  synthetic DNA construct
REFERENCE   1  (bases 1 to 11241)
  TITLE     Tunable and temporal expression control of Cas9
REFERENCE   2  (bases 1 to 11241)
  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"
COMMENT     Sequence Label: XClone_Cas9_KRAB_T2A_BFP
FEATURES             Location/Qualifiers
     source          1..11241
                     /mol_type="other DNA"
                     /organism="synthetic DNA construct"
     promoter        9..376
                     /label=TRE3GS promoter
                     /note="3rd-generation Tet-responsive promoter that can be 
                     activated by binding of Tet-On(R) 3G, modified to eliminate
                     binding sites for endogenous mammalian transcription 
                     factors"
     protein_bind    17..35
                     /gene="tetO"
                     /label=tet operator
                     /bound_moiety="tetracycline repressor TetR"
                     /note="bacterial operator O2 for the tetR and tetA genes"
     protein_bind    53..71
                     /gene="tetO"
                     /label=tet operator
                     /bound_moiety="tetracycline repressor TetR"
                     /note="bacterial operator O2 for the tetR and tetA genes"
     protein_bind    89..107
                     /gene="tetO"
                     /label=tet operator
                     /bound_moiety="tetracycline repressor TetR"
                     /note="bacterial operator O2 for the tetR and tetA genes"
     protein_bind    125..143
                     /gene="tetO"
                     /label=tet operator
                     /bound_moiety="tetracycline repressor TetR"
                     /note="bacterial operator O2 for the tetR and tetA genes"
     protein_bind    161..179
                     /gene="tetO"
                     /label=tet operator
                     /bound_moiety="tetracycline repressor TetR"
                     /note="bacterial operator O2 for the tetR and tetA genes"
     protein_bind    197..215
                     /gene="tetO"
                     /label=tet operator
                     /bound_moiety="tetracycline repressor TetR"
                     /note="bacterial operator O2 for the tetR and tetA genes"
     protein_bind    233..251
                     /gene="tetO"
                     /label=tet operator
                     /bound_moiety="tetracycline repressor TetR"
                     /note="bacterial operator O2 for the tetR and tetA genes"
     CDS             398..445
                     /codon_start=1
                     /product="bipartite nuclear localization signal from 
                     nucleoplasmin"
                     /label=nucleoplasmin NLS
                     /translation="KRPAATKKAGQAKKKK"
     CDS             join(446..469,470..472,473..3028,3029..3031,3032..4546)
                     /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 N863A mutation in the HNH 
                     catalytic domain"
                     /translation="DKKYSIGLAIGTNSVGWAVITDEYKVPSKKFKVLGNTDRHSIKKN
                     LIGALLFDSGETAEATRLKRTARRRYTRRKNRICYLQEIFSNEMAKVDDSFFHRLEESF
                     LVEEDKKHERHPIFGNIVDEVAYHEKYPTIYHLRKKLVDSTDKADLRLIYLALAHMIKF
                     RGHFLIEGDLNPDNSDVDKLFIQLVQTYNQLFEENPINASGVDAKAILSARLSKSRRLE
                     NLIAQLPGEKKNGLFGNLIALSLGLTPNFKSNFDLAEDAKLQLSKDTYDDDLDNLLAQI
                     GDQYADLFLAAKNLSDAILLSDILRVNTEITKAPLSASMIKRYDEHHQDLTLLKALVRQ
                     QLPEKYKEIFFDQSKNGYAGYIDGGASQEEFYKFIKPILEKMDGTEELLVKLNREDLLR
                     KQRTFDNGSIPHQIHLGELHAILRRQEDFYPFLKDNREKIEKILTFRIPYYVGPLARGN
                     SRFAWMTRKSEETITPWNFEEVVDKGASAQSFIERMTNFDKNLPNEKVLPKHSLLYEYF
                     TVYNELTKVKYVTEGMRKPAFLSGEQKKAIVDLLFKTNRKVTVKQLKEDYFKKIECFDS
                     VEISGVEDRFNASLGTYHDLLKIIKDKDFLDNEENEDILEDIVLTLTLFEDREMIEERL
                     KTYAHLFDDKVMKQLKRRRYTGWGRLSRKLINGIRDKQSGKTILDFLKSDGFANRNFMQ
                     LIHDDSLTFKEDIQKAQVSGQGDSLHEHIANLAGSPAIKKGILQTVKVVDELVKVMGRH
                     KPENIVIEMARENQTTQKGQKNSRERMKRIEEGIKELGSQILKEHPVENTQLQNEKLYL
                     YYLQNGRDMYVDQELDINRLSDYDVDHIVPQSFLKDDSIDNKVLTRSDKARGKSDNVPS
                     EEVVKKMKNYWRQLLNAKLITQRKFDNLTKAERGGLSELDKAGFIKRQLVETRQITKHV
                     AQILDSRMNTKYDENDKLIREVKVITLKSKLVSDFRKDFQFYKVREINNYHHAHDAYLN
                     AVVGTALIKKYPKLESEFVYGDYKVYDVRKMIAKSEQEIGKATAKYFFYSNIMNFFKTE
                     ITLANGEIRKRPLIETNGETGEIVWDKGRDFATVRKVLSMPQVNIVKKTEVQTGGFSKE
                     SILPKRNSDKLIARKKDWDPKKYGGFDSPTVAYSVLVVAKVEKGKSKKLKSVKELLGIT
                     IMERSSFEKNPIDFLEAKGYKEVKKDLIIKLPKYSLFELENGRKRMLASAGELQKGNEL
                     ALPSKYVNFLYLASHYEKLKGSPEDNEQKQLFVEQHKHYLDEIIEQISEFSKRVILADA
                     NLDKVLSAYNKHRDKPIREQAENIIHLFTLTNLGAPAAFKYFDTTIDRKRYTSTKEVLD
                     ATLIHQSITGLYETRIDLSQLGGD"
     CDS             4601..4621
                     /codon_start=1
                     /product="nuclear localization signal of SV40 (simian virus
                     40) large T antigen"
                     /label=SV40 NLS
                     /translation="PKKKRKV"
     CDS             5036..5230
                     /codon_start=1
                     /product="Kruppel-associated box (KRAB) transcriptional 
                     repression domain from the human zinc finger protein ZNF10 
                     (Margolin et al., 1994)"
                     /label=KRAB
                     /translation="RTLVTFKDVFVDFTREEWKLLDTAQQIVYRNVMLENYKNLVSLGY
                     QLTKPDVILRLEKGEEPWLV"
     CDS             5246..5299
                     /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             5312..6010
                     /codon_start=1
                     /product="monomeric blue fluorescent protein"
                     /label=TagBFP
                     /note="mammalian codon-optimized"
                     /translation="MSELIKENMHMKLYMEGTVDNHHFKCTSEGEGKPYEGTQTMRIKV
                     VEGGPLPFAFDILATSFLYGSKTFINHTQGIPDFFKQSFPEGFTWERVTTYEDGGVLTA
                     TQDTSLQDGCLIYNVKIRGVNFTSNGPVMQKKTLGWEAFTETLYPADGGLEGRNDMALK
                     LVGGSHLIANIKTTYRSKKPAKNLKMPGVYYVDYRLERIKEANNETYVEQHEVAVARYC
                     DLPSKLGHKLN"
     polyA_signal    6203..6337
                     /label=SV40 poly(A) signal
                     /note="SV40 polyadenylation signal"
     repeat_region   6613..6647
                     /label=piggyBac left (5') inverted repeat
                     /note="piggyBac transposon-specific inverted terminal 
                     repeat sequence (ITR)"
     primer_bind     complement(6658..6675)
                     /label=M13 Forward
                     /note="In lacZ gene. Also called M13-F20 or M13 (-21) 
                     Forward"
     primer_bind     complement(6658..6674)
                     /label=M13 fwd
                     /note="common sequencing primer, one of multiple similar 
                     variants"
     primer_bind     complement(6667..6689)
                     /label=M13/pUC Forward
                     /note="In lacZ gene"
     primer_bind     complement(6883..6902)
                     /label=pRS-marker
                     /note="pRS vectors, use to sequence yeast selectable 
                     marker"
     primer_bind     7002..7024
                     /label=pGEX 3'
                     /note="pGEX vectors, reverse primer"
     primer_bind     complement(7062..7080)
                     /label=pBRforEco
                     /note="pBR322 vectors, upsteam of EcoRI site, forward 
                     primer"
     promoter        7148..7252
                     /gene="bla"
                     /label=AmpR promoter
     CDS             join(7253..7321,7322..8113)
                     /codon_start=1
                     /gene="bla"
                     /product="beta-lactamase"
                     /label=AmpR
                     /note="confers resistance to ampicillin, carbenicillin, and
                     related antibiotics"
                     /translation="MSIQHFRVALIPFFAAFCLPVFAHPETLVKVKDAEDQLGARVGYI
                     ELDLNSGKILESFRPEERFPMMSTFKVLLCGAVLSRIDAGQEQLGRRIHYSQNDLVEYS
                     PVTEKHLTDGMTVRELCSAAITMSDNTAANLLLTTIGGPKELTAFLHNMGDHVTRLDRW
                     EPELNEAIPNDERDTTMPVAMATTLRKLLTGELLTLASRQQLIDWMEADKVAGPLLRSA
                     LPAGWFIADKSGAGERGSRGIIAALGPDGKPSRIVVIYTTGSQATMDERNRQIAEIGAS
                     LIKHW"
     primer_bind     complement(7471..7490)
                     /label=Amp-R
                     /note="Ampicillin resistance gene, reverse primer"
     rep_origin      8284..8872
                     /direction=RIGHT
                     /label=ori
                     /note="high-copy-number ColE1/pMB1/pBR322/pUC origin of 
                     replication"
     primer_bind     8773..8792
                     /label=pBR322ori-F
                     /note="pBR322 origin, forward primer"
     primer_bind     9026..9043
                     /label=L4440
                     /note="L4440 vector, forward primer"
     protein_bind    9160..9181
                     /label=CAP binding site
                     /bound_moiety="E. coli catabolite activator protein"
                     /note="CAP binding activates transcription in the presence 
                     of cAMP."
     promoter        join(9196..9201,9202..9219,9220..9226)
                     /label=lac promoter
                     /note="promoter for the E. coli lac operon"
     protein_bind    9234..9250
                     /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)."
     primer_bind     9239..9261
                     /label=M13/pUC Reverse
                     /note="In lacZ gene"
     primer_bind     9258..9274
                     /label=M13 rev
                     /note="common sequencing primer, one of multiple similar 
                     variants"
     primer_bind     9258..9274
                     /label=M13 Reverse
                     /note="In lacZ gene. Also called M13-rev"
     repeat_region   9300..9362
                     /label=piggyBac right (3') inverted repeat
                     /note="piggyBac transposon-specific inverted terminal 
                     repeat sequence (ITR)"
     polyA_signal    9538..9619
                     /label=SV40 poly(A) signal
                     /note="SV40 polyadenylation signal"
     CDS             complement(9736..10134)
                     /codon_start=1
                     /gene="Aspergillus terreus BSD"
                     /product="blasticidin S deaminase"
                     /label=BSD
                     /note="confers resistance to blasticidin"
                     /translation="MAKPLSQEESTLIERATATINSIPISEDYSVASAALSSDGRIFTG
                     VNVYHFTGGPCAELVVLGTAAAAAAGNLTCIVAIGNENRGILSPCGRCRQVLLDLHPGI
                     KAIVKDSDGQPTAVGIRELLPSGYVWEG"
     CDS             complement(10135..10191)
                     /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             complement(10201..10944)
                     /codon_start=1
                     /product="modified rtTA protein that binds tightly to 
                     promoters containing the tet operator in the presence of 
                     doxycycline"
                     /label=Tet-On(R) 3G
                     /translation="MSRLDKSKVINSALELLNGVGIEGLTTRKLAQKLGVEQPTLYWHV
                     KNKRALLDALPIEMLDRHHTHSCPLEGESWQDFLRNNAKSYRCALLSHRDGAKVHLGTR
                     PTEKQYETLENQLAFLCQQGFSLENALYALSAVGHFTLGCVLEEQEHQVAKEERETPTT
                     DSMPPLLKQAIELFDRQGAEPAFLFGLELIICGLEKQLKCESGGPTDALDDFDLDMLPA
                     DALDDFDLDMLPADALDDFDLDMLPG"
     CDS             complement(10945..10968)
                     /codon_start=1
                     /product="FLAG(R) epitope tag, followed by an enterokinase 
                     cleavage site"
                     /label=FLAG
                     /translation="DYKDDDDK"
     regulatory      10968..10977
                     /label=Kozak sequence
                     /note="vertebrate consensus sequence for strong initiation 
                     of translation (Kozak, 1987)"
                     /regulatory_class="other"
     promoter        complement(10997..11208)
                     /label=EF-1-alpha core promoter
                     /note="core promoter for human elongation factor 
                     EF-1-alpha"
ORIGIN
        1 atcacctcga gtttactccc tatcagtgat agagaacgta tgaagagttt actccctatc
       61 agtgatagag aacgtatgca gactttactc cctatcagtg atagagaacg tataaggagt
      121 ttactcccta tcagtgatag agaacgtatg accagtttac tccctatcag tgatagagaa
      181 cgtatctaca gtttactccc tatcagtgat agagaacgta tatccagttt actccctatc
      241 agtgatagag aacgtataag ctttgcttat gtaaaccagg gcgcctataa aagagtgctg
      301 attttttgag taaacttcaa ttccacaaca cttttgtctt ataccaactt tccgtaccac
      361 ttcctaccct cgtaaaggta cccgtacggc caccatgaaa aggccggcgg ccacgaaaaa
      421 ggccggccag gcaaaaaaga aaaaggacaa gaagtacagc atcggcctgg ccatcggcac
      481 caactctgtg ggctgggccg tgatcaccga cgagtacaag gtgcccagca agaaattcaa
      541 ggtgctgggc aacaccgacc ggcacagcat caagaagaac ctgatcggag ccctgctgtt
      601 cgacagcggc gaaacagccg aggccacccg gctgaagaga accgccagaa gaagatacac
      661 cagacggaag aaccggatct gctatctgca agagatcttc agcaacgaga tggccaaggt
      721 ggacgacagc ttcttccaca gactggaaga gtccttcctg gtggaagagg ataagaagca
      781 cgagcggcac cccatcttcg gcaacatcgt ggacgaggtg gcctaccacg agaagtaccc
      841 caccatctac cacctgagaa agaaactggt ggacagcacc gacaaggccg acctgcggct
      901 gatctatctg gccctggccc acatgatcaa gttccggggc cacttcctga tcgagggcga
      961 cctgaacccc gacaacagcg acgtggacaa gctgttcatc cagctggtgc agacctacaa
     1021 ccagctgttc gaggaaaacc ccatcaacgc cagcggcgtg gacgccaagg ccatcctgtc
     1081 tgccagactg agcaagagca gacggctgga aaatctgatc gcccagctgc ccggcgagaa
     1141 gaagaatggc ctgttcggca acctgattgc cctgagcctg ggcctgaccc ccaacttcaa
     1201 gagcaacttc gacctggccg aggatgccaa actgcagctg agcaaggaca cctacgacga
     1261 cgacctggac aacctgctgg cccagatcgg cgaccagtac gccgacctgt ttctggccgc
     1321 caagaacctg tccgacgcca tcctgctgag cgacatcctg agagtgaaca ccgagatcac
     1381 caaggccccc ctgagcgcct ctatgatcaa gagatacgac gagcaccacc aggacctgac
     1441 cctgctgaaa gctctcgtgc ggcagcagct gcctgagaag tacaaagaga ttttcttcga
     1501 ccagagcaag aacggctacg ccggctacat tgacggcgga gccagccagg aagagttcta
     1561 caagttcatc aagcccatcc tggaaaagat ggacggcacc gaggaactgc tcgtgaagct
     1621 gaacagagag gacctgctgc ggaagcagcg gaccttcgac aacggcagca tcccccacca
     1681 gatccacctg ggagagctgc acgccattct gcggcggcag gaagattttt acccattcct
     1741 gaaggacaac cgggaaaaga tcgagaagat cctgaccttc cgcatcccct actacgtggg
     1801 ccctctggcc aggggaaaca gcagattcgc ctggatgacc agaaagagcg aggaaaccat
     1861 caccccctgg aacttcgagg aagtggtgga caagggcgct tccgcccaga gcttcatcga
     1921 gcggatgacc aacttcgata agaacctgcc caacgagaag gtgctgccca agcacagcct
     1981 gctgtacgag tacttcaccg tgtataacga gctgaccaaa gtgaaatacg tgaccgaggg
     2041 aatgagaaag cccgccttcc tgagcggcga gcagaaaaag gccatcgtgg acctgctgtt
     2101 caagaccaac cggaaagtga ccgtgaagca gctgaaagag gactacttca agaaaatcga
     2161 gtgcttcgac tccgtggaaa tctccggcgt ggaagatcgg ttcaacgcct ccctgggcac
     2221 ataccacgat ctgctgaaaa ttatcaagga caaggacttc ctggacaatg aggaaaacga
     2281 ggacattctg gaagatatcg tgctgaccct gacactgttt gaggacagag agatgatcga
     2341 ggaacggctg aaaacctatg cccacctgtt cgacgacaaa gtgatgaagc agctgaagcg
     2401 gcggagatac accggctggg gcaggctgag ccggaagctg atcaacggca tccgggacaa
     2461 gcagtccggc aagacaatcc tggatttcct gaagtccgac ggcttcgcca acagaaactt
     2521 catgcagctg atccacgacg acagcctgac ctttaaagag gacatccaga aagcccaggt
     2581 gtccggccag ggcgatagcc tgcacgagca cattgccaat ctggccggca gccccgccat
     2641 taagaagggc atcctgcaga cagtgaaggt ggtggacgag ctcgtgaaag tgatgggccg
     2701 gcacaagccc gagaacatcg tgatcgaaat ggccagagag aaccagacca cccagaaggg
     2761 acagaagaac agccgcgaga gaatgaagcg gatcgaagag ggcatcaaag agctgggcag
     2821 ccagatcctg aaagaacacc ccgtggaaaa cacccagctg cagaacgaga agctgtacct
     2881 gtactacctg cagaatgggc gggatatgta cgtggaccag gaactggaca tcaaccggct
     2941 gtccgactac gatgtggacc acatcgtgcc tcagagcttt ctgaaggacg actccatcga
     3001 caacaaggtg ctgaccagaa gcgacaaggc ccggggcaag agcgacaacg tgccctccga
     3061 agaggtcgtg aagaagatga agaactactg gcggcagctg ctgaacgcca agctgattac
     3121 ccagagaaag ttcgacaatc tgaccaaggc cgagagaggc ggcctgagcg aactggataa
     3181 ggccggcttc atcaagagac agctggtgga aacccggcag atcacaaagc acgtggcaca
     3241 gatcctggac tcccggatga acactaagta cgacgagaat gacaagctga tccgggaagt
     3301 gaaagtgatc accctgaagt ccaagctggt gtccgatttc cggaaggatt tccagtttta
     3361 caaagtgcgc gagatcaaca actaccacca cgcccacgac gcctacctga acgccgtcgt
     3421 gggaaccgcc ctgatcaaaa agtaccctaa gctggaaagc gagttcgtgt acggcgacta
     3481 caaggtgtac gacgtgcgga agatgatcgc caagagcgag caggaaatcg gcaaggctac
     3541 cgccaagtac ttcttctaca gcaacatcat gaactttttc aagaccgaga ttaccctggc
     3601 caacggcgag atccggaagc ggcctctgat cgagacaaac ggcgaaaccg gggagatcgt
     3661 gtgggataag ggccgggatt ttgccaccgt gcggaaagtg ctgagcatgc cccaagtgaa
     3721 tatcgtgaaa aagaccgagg tgcagacagg cggcttcagc aaagagtcta tcctgcccaa
     3781 gaggaacagc gataagctga tcgccagaaa gaaggactgg gaccctaaga agtacggcgg
     3841 cttcgacagc cccaccgtgg cctattctgt gctggtggtg gccaaagtgg aaaagggcaa
     3901 gtccaagaaa ctgaagagtg tgaaagagct gctggggatc accatcatgg aaagaagcag
     3961 cttcgagaag aatcccatcg actttctgga agccaagggc tacaaagaag tgaaaaagga
     4021 cctgatcatc aagctgccta agtactccct gttcgagctg gaaaacggcc ggaagagaat
     4081 gctggcctct gccggcgaac tgcagaaggg aaacgaactg gccctgccct ccaaatatgt
     4141 gaacttcctg tacctggcca gccactatga gaagctgaag ggctcccccg aggataatga
     4201 gcagaaacag ctgtttgtgg aacagcacaa gcactacctg gacgagatca tcgagcagat
     4261 cagcgagttc tccaagagag tgatcctggc cgacgctaat ctggacaaag tgctgtccgc
     4321 ctacaacaag caccgggata agcccatcag agagcaggcc gagaatatca tccacctgtt
     4381 taccctgacc aatctgggag cccctgccgc cttcaagtac tttgacacca ccatcgaccg
     4441 gaagaggtac accagcacca aagaggtgct ggacgccacc ctgatccacc agagcatcac
     4501 cggcctgtac gagacacgga tcgacctgtc tcagctggga ggcgacagcg ctggaggagg
     4561 tggaagcgga ggaggaggaa gcggaggagg aggtagcgga cctaagaaaa agaggaaggt
     4621 ggcggccgct ggatccatga acatccagat gctgctggag gccgctgact acctggaacg
     4681 gagagagcgc gaagccgagc acggatatgc ttcaatgctg cccggaagcg gcatgaatat
     4741 tcagatgctg ctggaggctg ctgattacct ggaaaggcgc gaacgggagg ccgaacatgg
     4801 ctatgcttcc atgctgcctg ggtctggaat gaatatccaa atgctgctgg aggcagccga
     4861 ttacctggaa cggagagaaa gagaagccga gcacggatac gccagcatgc tgccaggcag
     4921 cgggatgaac atacaaatgc tgctggaggc tgccgattac ctggagaggc gcgagagaga
     4981 agctgaacat ggctatgcct ctatgctgcc cagtgggcag ggaggaggtg gaagcagaac
     5041 actggttacg ttcaaggacg tgtttgtgga ctttacacgt gaggagtgga aattgctgga
     5101 tactgcgcaa caaattgtgt atcgaaatgt catgcttgag aattacaaga acctcgtcag
     5161 tctcggatac cagttgacga aaccggatgt gatccttagg ctcgaaaagg gggaagaacc
     5221 ttggctggta ggtaccggat ccggagaggg cagaggaagt ctgctaacat gcggtgacgt
     5281 cgaggagaat cctggcccac cggtcgccac catgagcgag ctgattaagg agaacatgca
     5341 catgaagctg tacatggagg gcaccgtgga caaccatcac ttcaagtgca catccgaggg
     5401 cgaaggcaag ccctacgagg gcacccagac catgagaatc aaggtggtcg agggcggccc
     5461 tctccccttc gccttcgaca tcctggctac tagcttcctc tacggcagca agaccttcat
     5521 caaccacacc cagggcatcc ccgacttctt caagcagtcc ttccctgagg gcttcacatg
     5581 ggagagagtc accacatacg aagacggggg cgtgctgacc gctacccagg acaccagcct
     5641 ccaggacggc tgcctcatct acaacgtcaa gatcagaggg gtgaacttca catccaacgg
     5701 ccctgtgatg cagaagaaaa cactcggctg ggaggccttc accgagacgc tgtaccccgc
     5761 tgacggcggc ctggaaggca gaaacgacat ggccctgaag ctcgtgggcg ggagccatct
     5821 gatcgcaaac atcaagacca catatagatc caagaaaccc gctaagaacc tcaagatgcc
     5881 tggcgtctac tatgtggact acagactgga aagaatcaag gaggccaaca acgagaccta
     5941 cgtcgagcag cacgaggtgg cagtggccag atactgcgac ctccctagca aactggggca
     6001 caagcttaat ggatccggct aaactagtag accacctccc ctgcgagcta agctggacag
     6061 ccaatgacgg gtaagagagt gacatttttc actaacctaa gacaggaggg ccgtcagagc
     6121 tactgcctaa tccaaagacg ggtaaaagtg ataaaaatgt atcactccaa cctaagacag
     6181 gcgcagcttc cgagggattt gagatccaga catgataaga tacattgatg agtttggaca
     6241 aaccaaaact agaatgcagt gaaaaaaatg ccttatttgt gaaatttgtg atgctattgc
     6301 cttatttgta accattataa gctgcaataa acaagtttga tatctataac aagaaaatat
     6361 atatataata agttatcacg taagtagaac atgaaataac aatataatta tcgtatgagt
     6421 taaatcttaa aagtcacgta aaagataatc atgcgtcatt ttgactcacg cggtcgttat
     6481 agttcaaaat cagtgacact taccgcattg acaagcacgc ctcacgggag ctccaagcgg
     6541 cgactgagat gtcctaaatg cacagcgacg gattcgcgct atttagaaag agagagcaat
     6601 atttcaagaa tgcatgcgtc aattttacgc agactatctt tctagggtta agaattcact
     6661 ggccgtcgtt ttacaacgtc gtgactggga aaaccctggc gttacccaac ttaatcgcct
     6721 tgcagcacat ccccctttcg ccagctggcg taatagcgaa gaggcccgca ccgatcgccc
     6781 ttcccaacag ttgcgcagcc tgaatggcga atggcgcctg atgcggtatt ttctccttac
     6841 gcatctgtgc ggtatttcac accgcatatg gtgcactctc agtacaatct gctctgatgc
     6901 cgcatagtta agccagcccc gacacccgcc aacacccgct gacgcgccct gacgggcttg
     6961 tctgctcccg gcatccgctt acagacaagc tgtgaccgtc tccgggagct gcatgtgtca
     7021 gaggttttca ccgtcatcac cgaaacgcgc gagacgaaag ggcctcgtga tacgcctatt
     7081 tttataggtt aatgtcatga taataatggt ttcttagacg tcaggtggca cttttcgggg
     7141 aaatgtgcgc ggaaccccta tttgtttatt tttctaaata cattcaaata tgtatccgct
     7201 catgagacaa taaccctgat aaatgcttca ataatattga aaaaggaaga gtatgagtat
     7261 tcaacatttc cgtgtcgccc ttattccctt ttttgcggca ttttgccttc ctgtttttgc
     7321 tcacccagaa acgctggtga aagtaaaaga tgctgaagat cagttgggtg cacgagtggg
     7381 ttacatcgaa ctggatctca acagcggtaa gatccttgag agttttcgcc ccgaagaacg
     7441 ttttccaatg atgagcactt ttaaagttct gctatgtggc gcggtattat cccgtattga
     7501 cgccgggcaa gagcaactcg gtcgccgcat acactattct cagaatgact tggttgagta
     7561 ctcaccagtc acagaaaagc atcttacgga tggcatgaca gtaagagaat tatgcagtgc
     7621 tgccataacc atgagtgata acactgcggc caacttactt ctgacaacga tcggaggacc
     7681 gaaggagcta accgcttttt tgcacaacat gggggatcat gtaactcgcc ttgatcgttg
     7741 ggaaccggag ctgaatgaag ccataccaaa cgacgagcgt gacaccacga tgcctgtagc
     7801 aatggcaaca acgttgcgca aactattaac tggcgaacta cttactctag cttcccggca
     7861 acaattaata gactggatgg aggcggataa agttgcagga ccacttctgc gctcggccct
     7921 tccggctggc tggtttattg ctgataaatc tggagccggt gagcgtgggt ctcgcggtat
     7981 cattgcagca ctggggccag atggtaagcc ctcccgtatc gtagttatct acacgacggg
     8041 gagtcaggca actatggatg aacgaaatag acagatcgct gagataggtg cctcactgat
     8101 taagcattgg taactgtcag accaagttta ctcatatata ctttagattg atttaaaact
     8161 tcatttttaa tttaaaagga tctaggtgaa gatccttttt gataatctca tgaccaaaat
     8221 cccttaacgt gagttttcgt tccactgagc gtcagacccc gtagaaaaga tcaaaggatc
     8281 ttcttgagat cctttttttc tgcgcgtaat ctgctgcttg caaacaaaaa aaccaccgct
     8341 accagcggtg gtttgtttgc cggatcaaga gctaccaact ctttttccga aggtaactgg
     8401 cttcagcaga gcgcagatac caaatactgt tcttctagtg tagccgtagt taggccacca
     8461 cttcaagaac tctgtagcac cgcctacata cctcgctctg ctaatcctgt taccagtggc
     8521 tgctgccagt ggcgataagt cgtgtcttac cgggttggac tcaagacgat agttaccgga
     8581 taaggcgcag cggtcgggct gaacgggggg ttcgtgcaca cagcccagct tggagcgaac
     8641 gacctacacc gaactgagat acctacagcg tgagctatga gaaagcgcca cgcttcccga
     8701 agggagaaag gcggacaggt atccggtaag cggcagggtc ggaacaggag agcgcacgag
     8761 ggagcttcca gggggaaacg cctggtatct ttatagtcct gtcgggtttc gccacctctg
     8821 acttgagcgt cgatttttgt gatgctcgtc aggggggcgg agcctatgga aaaacgccag
     8881 caacgcggcc tttttacggt tcctggcctt ttgctggcct tttgctcaca tgttctttcc
     8941 tgcgttatcc cctgattctg tggataaccg tattaccgcc tttgagtgag ctgataccgc
     9001 tcgccgcagc cgaacgaccg agcgcagcga gtcagtgagc gaggaagcgg aagagcgccc
     9061 aatacgcaaa ccgcctctcc ccgcgcgttg gccgattcat taatgcagct ggcacgacag
     9121 gtttcccgac tggaaagcgg gcagtgagcg caacgcaatt aatgtgagtt agctcactca
     9181 ttaggcaccc caggctttac actttatgct tccggctcgt atgttgtgtg gaattgtgag
     9241 cggataacaa tttcacacag gaaacagcta tgaccatgat tacgccaagg tcgacttaac
     9301 cctagaaaga taatcatatt gtgacgtacg ttaaagataa tcatgcgtaa aattgacgca
     9361 tgtgttttat cggtctgtat atcgaggttt atttattaat ttgaatagat attaagtttt
     9421 attatattta cacttacata ctaataataa attcaacaaa caatttattt atgtttattt
     9481 atttattaaa aaaaaacaaa aactcaaaat ttcttctata aagtaacaaa acttttagca
     9541 gtgaaaaaaa tgctttattt gtgaaatttg tgatgctatt gctttatttg taaccattat
     9601 aagctgcaat aaacaagtta acaacaacaa ttgcattcat tttatgtttc aggttcaggg
     9661 ggaggtgtgg gaggtttttt aaagcaagta aaacctctac aaatgtggta tggctgatta
     9721 tgatcctctg gagatttagc cctcccacac ataaccagag ggcagcaatt cacgaatccc
     9781 aactgccgtc ggctgtccat cactgtcctt cactatggct ttgatcccag gatgcagatc
     9841 gagaagcacc tgtcggcacc gtccgcaggg gctcaagatg cccctgttct catttccgat
     9901 cgcgacgata caagtcaggt tgccagctgc cgcagcagca gcagtgccca gcaccacgag
     9961 ttctgcacaa ggtcccccag taaaatgata tacattgaca ccagtgaaga tgcggccgtc
    10021 gctagagaga gctgcgctgg cgacgctgta gtcttcagag atggggatgc tgttgattgt
    10081 agccgttgct ctttcaatga gggtggattc ttcttgagac aaaggcttgg ccatggggcc
    10141 ggggttctcc tccacgtcgc cggcctgctt cagcaggctg aagttggtgg cgccgctgcc
    10201 cccggggagc atgtcaaggt caaaatcgtc aagagcgtca gcaggcagca tatcaaggtc
    10261 aaagtcgtca agggcatcgg ctgggagcat gtctaagtca aaatcgtcaa gggcgtcggt
    10321 cggcccgccg ctttcgcact ttagctgttt ctccaggcca catatgatta gttccaggcc
    10381 gaaaaggaag gcaggttcgg ctccctgccg gtcgaacagc tcaattgctt gtttcagaag
    10441 tgggggcata gaatcggtgg taggtgtctc tctttcctct tttgctactt gatgctcctg
    10501 ttcctccaat acgcagccca gtgtaaagtg gcccacggcg gacagagcgt acagtgcgtt
    10561 ctccagggag aagccttgct gacacaggaa cgcgagctga ttttccaggg tttcgtactg
    10621 tttctctgtt gggcgggtgc cgagatgcac tttagccccg tcgcgatgtg agaggagagc
    10681 acagcggtat gacttggcgt tgttccgcag aaagtcttgc catgactcgc cttccagggg
    10741 gcaggagtgg gtatgatgcc tgtccagcat ctcgattggc agggcatcga gcagggcccg
    10801 cttgttcttc acgtgccagt acagggtagg ctgctcaact cccagctttt gagcgagttt
    10861 ccttgtcgtc aggccttcga taccgactcc attgagtaat tccagagcag agtttatgac
    10921 tttgctcttg tccagtctag acatcttatc gtcatcgtct ttgtaatcca tggtggcgga
    10981 tcccgcgtca cgacacctgt gttctggcgg caaacccgtt gcgaaaaaga acgttcacgg
    11041 cgactactgc acttatatac ggttctcccc caccctcggg aaaaaggcgg agccagtaca
    11101 cgacatcact ttcccagttt accccgcgcc accttctcta ggcaccggtt caattgccga
    11161 cccctccccc caacttctcg gggactgtgg gcgatgtgcg ctctgcccac tgacgggcac
    11221 cggagccact cgagtggaat t
//
