LOCUS       sequence-388978-       11724 bp    DNA     circular SYN 15-DEC-2025
DEFINITION  (Empty Backbone) Inducible CRISPRi vector conferring hygromycin B 
            resistance, ready for insertion of gRNA targeting gene of interest 
            using SapI restriction sites..
ACCESSION   .
VERSION     .
KEYWORDS    .
SOURCE      synthetic DNA construct
  ORGANISM  synthetic DNA construct
REFERENCE   1  (bases 1 to 11724)
  AUTHORS   Czarnek M, Kochan J, Wawro M, Myrczek R, Bereta J
  TITLE     Construction of a Set of Novel Transposon Vectors for Efficient 
            Silencing of Protein and lncRNA Genes via CRISPR Interference.
  JOURNAL   Mol Biotechnol. 2023 Jan 28. doi: 10.1007/s12033-023-00675-5.
  PUBMED    36707469
REFERENCE   2  (bases 1 to 11724)
  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: "Mol 
            Biotechnol. 2023 Jan 28. doi: 10.1007/s12033-023-00675-5."
COMMENT     Sequence Label: pSBtet-Hyg-dCas9-KRAB-MeCP2_hU6-SapI
FEATURES             Location/Qualifiers
     source          1..11724
                     /mol_type="other DNA"
                     /organism="synthetic DNA construct"
     promoter        295..607
                     /label=tight TRE promoter
                     /note="Tet-responsive promoter PTight, consisting of seven 
                     tet operator sequences followed by the minimal CMV 
                     promoter"
     protein_bind    303..321
                     /gene="tetO"
                     /label=tet operator
                     /bound_moiety="tetracycline repressor TetR"
                     /note="bacterial operator O2 for the tetR and tetA genes"
     protein_bind    339..357
                     /gene="tetO"
                     /label=tet operator
                     /bound_moiety="tetracycline repressor TetR"
                     /note="bacterial operator O2 for the tetR and tetA genes"
     protein_bind    374..392
                     /gene="tetO"
                     /label=tet operator
                     /bound_moiety="tetracycline repressor TetR"
                     /note="bacterial operator O2 for the tetR and tetA genes"
     protein_bind    410..428
                     /gene="tetO"
                     /label=tet operator
                     /bound_moiety="tetracycline repressor TetR"
                     /note="bacterial operator O2 for the tetR and tetA genes"
     protein_bind    446..464
                     /gene="tetO"
                     /label=tet operator
                     /bound_moiety="tetracycline repressor TetR"
                     /note="bacterial operator O2 for the tetR and tetA genes"
     protein_bind    481..499
                     /gene="tetO"
                     /label=tet operator
                     /bound_moiety="tetracycline repressor TetR"
                     /note="bacterial operator O2 for the tetR and tetA genes"
     protein_bind    517..535
                     /gene="tetO"
                     /label=tet operator
                     /bound_moiety="tetracycline repressor TetR"
                     /note="bacterial operator O2 for the tetR and tetA genes"
     primer_bind     580..604
                     /label=LNCX
                     /note="Human CMV promoter, forward primer"
     regulatory      683..692
                     /label=Kozak sequence
                     /note="vertebrate consensus sequence for strong initiation 
                     of translation (Kozak, 1987)"
                     /regulatory_class="other"
     CDS             join(689..715,716..718,719..3202,3203..3208,3209..3274,
                     3275..3277,3278..4792)
                     /codon_start=1
                     /product="catalytically dead mutant of the Cas9 
                     endonuclease from the Streptococcus pyogenes Type II 
                     CRISPR/Cas system"
                     /label=Cas9m4
                     /note="RNA-guided DNA-binding protein that lacks 
                     endonuclease activity due to the mutations D10A, D839A, 
                     H840A, and N863A (Mali et al., 2013)"
                     /translation="MDKKYSIGLAIGTNSVGWAVITDEYKVPSKKFKVLGNTDRHSIKK
                     NLIGALLFDSGETAEATRLKRTARRRYTRRKNRICYLQEIFSNEMAKVDDSFFHRLEES
                     FLVEEDKKHERHPIFGNIVDEVAYHEKYPTIYHLRKKLVDSTDKADLRLIYLALAHMIK
                     FRGHFLIEGDLNPDNSDVDKLFIQLVQTYNQLFEENPINASGVDAKAILSARLSKSRRL
                     ENLIAQLPGEKKNGLFGNLIALSLGLTPNFKSNFDLAEDAKLQLSKDTYDDDLDNLLAQ
                     IGDQYADLFLAAKNLSDAILLSDILRVNTEITKAPLSASMIKRYDEHHQDLTLLKALVR
                     QQLPEKYKEIFFDQSKNGYAGYIDGGASQEEFYKFIKPILEKMDGTEELLVKLNREDLL
                     RKQRTFDNGSIPHQIHLGELHAILRRQEDFYPFLKDNREKIEKILTFRIPYYVGPLARG
                     NSRFAWMTRKSEETITPWNFEEVVDKGASAQSFIERMTNFDKNLPNEKVLPKHSLLYEY
                     FTVYNELTKVKYVTEGMRKPAFLSGEQKKAIVDLLFKTNRKVTVKQLKEDYFKKIECFD
                     SVEISGVEDRFNASLGTYHDLLKIIKDKDFLDNEENEDILEDIVLTLTLFEDREMIEER
                     LKTYAHLFDDKVMKQLKRRRYTGWGRLSRKLINGIRDKQSGKTILDFLKSDGFANRNFM
                     QLIHDDSLTFKEDIQKAQVSGQGDSLHEHIANLAGSPAIKKGILQTVKVVDELVKVMGR
                     HKPENIVIEMARENQTTQKGQKNSRERMKRIEEGIKELGSQILKEHPVENTQLQNEKLY
                     LYYLQNGRDMYVDQELDINRLSDYDVAAIVPQSFLKDDSIDNKVLTRSDKARGKSDNVP
                     SEEVVKKMKNYWRQLLNAKLITQRKFDNLTKAERGGLSELDKAGFIKRQLVETRQITKH
                     VAQILDSRMNTKYDENDKLIREVKVITLKSKLVSDFRKDFQFYKVREINNYHHAHDAYL
                     NAVVGTALIKKYPKLESEFVYGDYKVYDVRKMIAKSEQEIGKATAKYFFYSNIMNFFKT
                     EITLANGEIRKRPLIETNGETGEIVWDKGRDFATVRKVLSMPQVNIVKKTEVQTGGFSK
                     ESILPKRNSDKLIARKKDWDPKKYGGFDSPTVAYSVLVVAKVEKGKSKKLKSVKELLGI
                     TIMERSSFEKNPIDFLEAKGYKEVKKDLIIKLPKYSLFELENGRKRMLASAGELQKGNE
                     LALPSKYVNFLYLASHYEKLKGSPEDNEQKQLFVEQHKHYLDEIIEQISEFSKRVILAD
                     ANLDKVLSAYNKHRDKPIREQAENIIHLFTLTNLGAPAAFKYFDTTIDRKRYTSTKEVL
                     DATLIHQSITGLYETRIDLSQLGGD"
     CDS             4805..4825
                     /codon_start=1
                     /product="nuclear localization signal of SV40 (simian virus
                     40) large T antigen"
                     /label=SV40 NLS
                     /translation="PKKKRKV"
     CDS             4886..5080
                     /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             5114..5134
                     /codon_start=1
                     /product="nuclear localization signal of SV40 (simian virus
                     40) large T antigen"
                     /label=SV40 NLS
                     /translation="PKKKRKV"
     CDS             5144..6001
                     /codon_start=1
                     /product="transcriptional repression domain from rat 
                     methyl-CpG-binding protein 2 (Nan et al., 1997)"
                     /label=MeCP2
                     /translation="VQVKRVLEKSPGKLLVKMPFQASPGGKGEGGGATTSAQVMVIKRP
                     GRKRKAEADPQAIPKKRGRKPGSVVAAAAAEAKKKAVKESSIRSVQETVLPIKKRKTRE
                     TVSIEVKEVVKPLLVSTLGEKSGKGLKTCKSPGRKSKESSPKGRSSSASSPPKKEHHHH
                     HHHAESPKAPMPLLPPPPPPEPQSSEDPISPPEPQDLSSSICKEEKMPRAGSLESDGCP
                     KEPAKTQPMVAAAATTTTTTTTTVAEKYKHRGEGERKDIVSSSMPRPNREEPVDSRTPV
                     TERVS"
     polyA_signal    6040..6161
                     /label=SV40 poly(A) signal
                     /note="SV40 polyadenylation signal"
     primer_bind     complement(6051..6070)
                     /label=EBV-rev
                     /note="SV40 polyA terminator, reverse primer"
     primer_bind     6105..6124
                     /label=SV40pA-R
                     /note="SV40 polyA, reverse primer"
     promoter        6255..6503
                     /label=U6 promoter
                     /note="RNA polymerase III promoter for human U6 snRNA 
                     (Domitrovich & Kunkel, 2003)"
     primer_bind     6255..6275
                     /label=hU6-F
                     /note="Human U6 promoter, forward primer"
     primer_bind     6426..6445
                     /label=LKO.1 5'
                     /note="Human U6 promoter, forward primer"
     misc_RNA        6522..6597
                     /label=gRNA scaffold
                     /note="guide RNA scaffold for the Streptococcus pyogenes 
                     CRISPR/Cas9 system"
     CDS             join(7244..7861,7862..7867,7868..7903,7904..7906,7907..7942,
                     7943..7945,7946..7981,7982..7987)
                     /codon_start=1
                     /product="improved tetracycline-controlled transactivator"
                     /label=rtTA-Advanced
                     /note="In the Tet-On(R) system, rtTA-Advanced binds to the 
                     Tet-responsive element and stimulates transcription only in
                     the presence of tetracycline or doxycycline."
                     /translation="MSRLDKSKVINGALELLNGVGIEGLTTRKLAQKLGVEQPTLYWHV
                     KNKRALLDALPIEMLDRHHTHFCPLEGESWQDFLRNNAKSFRCALLSHRDGAKVHLGTR
                     PTEKQYETLENQLAFLCQQGFSLENALYALSAVGHFTLGCVLEEQEHQVAKEERETPTT
                     DSMPPLLRQAIELFDRQGAEPAFLFGLELIICGLEKQLKCESGGPADALDDFDLDMLPA
                     DALDDFDLDMLPADALDDFDLDMLPG"
     CDS             7994..8050
                     /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             8051..9076
                     /codon_start=1
                     /gene="aph(4)-Ia"
                     /product="aminoglycoside phosphotransferase from E. coli"
                     /label=HygR
                     /note="confers resistance to hygromycin"
                     /translation="MKKPELTATSVEKFLIEKFDSVSDLMQLSEGEESRAFSFDVGGRG
                     YVLRVNSCADGFYKDRYVYRHFASAALPIPEVLDIGEFSESLTYCISRRAQGVMLQDLP
                     ETELPAVLQPVAEAMDAIAAADLSQTSGFGPFGPQGIGQYTTWRDFICAIADPHVYHWQ
                     TVMDDTVSASVAQALDELMLWAEDCPEVRHLVHADFGSNNVLTDNGRITAVIDWSEAMF
                     GDSQYEVANIFFWRPWLACMEQQTRYFERRHPELAGSPRLRAYMLRIGLDQLYQSLVDG
                     NFDDAAWAQGRCDAIVRSGAGTVGRTQIARRSAAVWTDGCVEVLADSGNRRPSTRPRAK
                     E"
     primer_bind     complement(9165..9182)
                     /label=BGH-rev
                     /note="Bovine growth hormone terminator, reverse primer. 
                     Also called BGH reverse"
     polyA_signal    9171..9395
                     /label=bGH poly(A) signal
                     /note="bovine growth hormone polyadenylation signal"
     primer_bind     complement(9721..9738)
                     /label=L4440
                     /note="L4440 vector, forward primer"
     rep_origin      complement(9892..10480)
                     /direction=LEFT
                     /label=ori
                     /note="high-copy-number ColE1/pMB1/pBR322/pUC origin of 
                     replication"
     primer_bind     complement(9972..9991)
                     /label=pBR322ori-F
                     /note="pBR322 origin, forward primer"
     CDS             complement(join(10651..11442,11443..11511))
                     /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     11274..11293
                     /label=Amp-R
                     /note="Ampicillin resistance gene, reverse primer"
     promoter        complement(11512..11616)
                     /gene="bla"
                     /label=AmpR promoter
ORIGIN
        1 ggatccctat acagttgaag tcggaagttt acatacactt aagttggagt cattaaaact
       61 cgtttttcaa ctactccaca aatttcttgt taacaaacaa tagttttggc aagtcagtta
      121 ggacatctac tttgtgcatg acacaagtca tttttccaac aattgtttac agacagatta
      181 tttcacttat aattcactgt atcacaattc cagtgggtca gaagtttaca tacactaagt
      241 tcgactcctc tgcagaatgc ggcgatgttt cggtaagggg tccgctatct agacgagttt
      301 actccctatc agtgatagag aacgtatgtc gagtttactc cctatcagtg atagagaacg
      361 atgtcgagtt tactccctat cagtgataga gaacgtatgt cgagtttact ccctatcagt
      421 gatagagaac gtatgtcgag tttactccct atcagtgata gagaacgtat gtcgagttta
      481 tccctatcag tgatagagaa cgtatgtcga gtttactccc tatcagtgat agagaacgta
      541 tgtcgattag gcgtgtacgg tgggcgccta taaaagcaga gctcgtttag tgaaccgtca
      601 gatcgcctgg agccaattcc aactcttttg ccttatacct actttccgta ccacttccta
      661 ccctcgaaag gcctctgagg ccgccaccat ggacaagaag tactccattg ggctcgctat
      721 cggcacaaac agcgtcggct gggccgtcat tacggacgag tacaaggtgc cgagcaaaaa
      781 attcaaagtt ctgggcaata ccgatcgcca cagcataaag aagaacctca ttggcgccct
      841 cctgttcgac tccggggaga cggccgaagc cacgcggctc aaaagaacag cacggcgcag
      901 atatacccgc agaaagaatc ggatctgcta cctgcaggag atctttagta atgagatggc
      961 taaggtggat gactctttct tccataggct ggaggagtcc tttttggtgg aggaggataa
     1021 aaagcacgag cgccacccaa tctttggcaa tatcgtggac gaggtggcgt accatgaaaa
     1081 gtacccaacc atatatcatc tgaggaagaa gcttgtagac agtactgata aggctgactt
     1141 gcggttgatc tatctcgcgc tggcgcatat gatcaaattt cggggacact tcctcatcga
     1201 gggggacctg aacccagaca acagcgatgt cgacaaactc tttatccaac tggttcagac
     1261 ttacaatcag cttttcgaag agaacccgat caacgcatcc ggagttgacg ccaaagcaat
     1321 cctgagcgct aggctgtcca aatcccggcg gctcgaaaac ctcatcgcac agctccctgg
     1381 ggagaagaag aacggcctgt ttggtaatct tatcgccctg tcactcgggc tgacccccaa
     1441 ctttaaatct aacttcgacc tggccgaaga tgccaagctt caactgagca aagacaccta
     1501 cgatgatgat ctcgacaatc tgctggccca gatcggcgac cagtacgcag accttttttt
     1561 ggcggcaaag aacctgtcag acgccattct gctgagtgat attctgcgag tgaacacgga
     1621 gatcaccaaa gctccgctga gcgctagtat gatcaagcgc tatgatgagc accaccaaga
     1681 cttgactttg ctgaaggccc ttgtcagaca gcaactgcct gagaagtaca aggaaatttt
     1741 cttcgatcag tctaaaaatg gctacgccgg atacattgac ggcggagcaa gccaggagga
     1801 attttacaaa tttattaagc ccatcttgga aaaaatggac ggcaccgagg agctgctggt
     1861 aaagcttaac agagaagatc tgttgcgcaa acagcgcact ttcgacaatg gaagcatccc
     1921 ccaccagatt cacctgggcg aactgcacgc tatcctcagg cggcaagagg atttctaccc
     1981 ctttttgaaa gataacaggg aaaagattga gaaaatcctc acatttcgga taccctacta
     2041 tgtaggcccc ctcgcccggg gaaattccag attcgcgtgg atgactcgca aatcagaaga
     2101 gaccatcact ccctggaact tcgaggaagt cgtggataag ggggcctctg cccagtcctt
     2161 catcgaaagg atgactaact ttgataaaaa tctgcctaac gaaaaggtgc ttcctaaaca
     2221 ctctctgctg tacgagtact tcacagttta taacgagctc accaaggtca aatacgtcac
     2281 agaagggatg agaaagccag cattcctgtc tggagagcag aagaaagcta tcgtggacct
     2341 cctcttcaag acgaaccgga aagttaccgt gaaacagctc aaagaagact atttcaaaaa
     2401 gattgaatgt ttcgactctg ttgaaatcag cggagtggag gatcgcttca acgcatccct
     2461 gggaacgtat cacgatctcc tgaaaatcat taaagacaag gacttcctgg acaatgagga
     2521 gaacgaggac attcttgagg acattgtcct cacccttacg ttgtttgaag atagggagat
     2581 gattgaagaa cgcttgaaaa cttacgctca tctcttcgac gacaaagtca tgaaacagct
     2641 caagaggcgc cgatatacag gatgggggcg gctgtcaaga aaactgatca atgggatccg
     2701 agacaagcag agtggaaaga caatcctgga ttttcttaag tccgatggat ttgccaaccg
     2761 gaacttcatg cagttgatcc atgatgactc tctcaccttt aaggaggaca tccagaaagc
     2821 acaagtttct ggccaggggg acagtcttca cgagcacatc gctaatcttg caggtagccc
     2881 agctatcaaa aagggaatac tgcagaccgt taaggtcgtg gatgaactcg tcaaagtaat
     2941 gggaaggcat aagcccgaga atatcgttat cgagatggcc cgagagaacc aaactaccca
     3001 gaagggacag aagaacagta gggaaaggat gaagaggatt gaagagggta taaaagaact
     3061 ggggtcccaa atccttaagg aacacccagt tgaaaacacc cagcttcaga atgagaagct
     3121 ctacctgtac tacctgcaga acggcaggga catgtacgtg gatcaggaac tggacatcaa
     3181 tcggctctcc gactacgacg tggctgctat cgtgccccag tcttttctca aagatgattc
     3241 tattgataat aaagtgttga caagatccga taaagctaga gggaagagtg ataacgtccc
     3301 ctcagaagaa gttgtcaaga aaatgaaaaa ttattggcgg cagctgctga acgccaaact
     3361 gatcacacaa cggaagttcg ataatctgac taaggctgaa cgaggtggcc tgtctgagtt
     3421 ggataaagcc ggcttcatca aaaggcagct tgttgagaca cgccagatca ccaagcacgt
     3481 ggcccaaatt ctcgattcac gcatgaacac caagtacgat gaaaatgaca aactgattcg
     3541 agaggtgaaa gttattactc tgaagtctaa gctggtctca gatttcagaa aggactttca
     3601 gttttataag gtgagagaga tcaacaatta ccaccatgcg catgatgcct acctgaatgc
     3661 agtggtaggc actgcactta tcaaaaaata tcccaagctt gaatctgaat ttgtttacgg
     3721 agactataaa gtgtacgatg ttaggaaaat gatcgcaaag tctgagcagg aaataggcaa
     3781 ggccaccgct aagtacttct tttacagcaa tattatgaat tttttcaaga ccgagattac
     3841 actggccaat ggagagattc ggaagcgacc acttatcgaa acaaacggag aaacaggaga
     3901 aatcgtgtgg gacaagggta gggatttcgc gacagtccgg aaggtcctgt ccatgccgca
     3961 ggtgaacatc gttaaaaaga ccgaagtaca gaccggaggc ttctccaagg aaagtatcct
     4021 cccgaaaagg aacagcgaca agctgatcgc acgcaaaaaa gattgggacc ccaagaaata
     4081 cggcggattc gattctccta cagtcgctta cagtgtactg gttgtggcca aagtggagaa
     4141 agggaagtct aaaaaactca aaagcgtcaa ggaactgctg ggcatcacaa tcatggagcg
     4201 atcaagcttc gaaaaaaacc ccatcgactt tctcgaggcg aaaggatata aagaggtcaa
     4261 aaaagacctc atcattaagc ttcccaagta ctctctcttt gagcttgaaa acggccggaa
     4321 acgaatgctc gctagtgcgg gcgagctgca gaaaggtaac gagctggcac tgccctctaa
     4381 atacgttaat ttcttgtatc tggccagcca ctatgaaaag ctcaaagggt ctcccgaaga
     4441 taatgagcag aagcagctgt tcgtggaaca acacaaacac taccttgatg agatcatcga
     4501 gcaaataagc gaattctcca aaagagtgat cctcgccgac gctaacctcg ataaggtgct
     4561 ttctgcttac aataagcaca gggataagcc catcagggag caggcagaaa acattatcca
     4621 cttgtttact ctgaccaact tgggcgcgcc tgcagccttc aagtacttcg acaccaccat
     4681 agacagaaag cggtacacct ctacaaagga ggtcctggac gccacactga ttcatcagtc
     4741 aattacgggg ctctatgaaa caagaatcga cctctctcag ctcggtggag acagcagggc
     4801 tgaccccaag aagaagagga aggtgagtgg tggaggaagt ggcgggtcag ggtcgatgga
     4861 cgcgaaatca cttacggcat ggtcgagaac actggttacg ttcaaggacg tgtttgtgga
     4921 ctttacacgt gaggagtgga aattgctgga tactgcgcaa caaattgtgt atcgaaatgt
     4981 catgcttgag aattacaaga acctcgtcag tctcggatac cagttgacga aaccggatgt
     5041 gatccttagg ctcgaaaagg gggaagaacc ttggctggta tcgggaggtg gttcgggtgg
     5101 ctctggatca agcccaaaga agaaacggaa ggtggaagcc tcagtgcagg tgaaaagggt
     5161 gctggaaaaa tcccccggca aactcctcgt gaagatgccc ttccaggctt cccctggcgg
     5221 aaaaggtgaa gggggtggcg caaccacatc tgcccaggtc atggtcatca agcgacctgg
     5281 aaggaaaaga aaggccgagg ctgaccctca ggccattcca aagaaacggg gacgcaagcc
     5341 agggtccgtg gtcgcagctg cagcagctga ggctaagaaa aaggcagtga aggaaagctc
     5401 catccgcagt gtgcaggaga ctgtcctgcc catcaagaag aggaagacta gggagaccgt
     5461 gtccatcgag gtcaaagaag tggtcaagcc cctgctcgtg tccaccctgg gcgaaaaatc
     5521 tggaaagggg ctcaaaacat gcaagtcacc tggacggaaa agcaaggagt ctagtccaaa
     5581 ggggcgctca agctccgctt ctagtccccc taaaaaggaa caccatcacc atcaccatca
     5641 cgccgagtct cctaaggctc ctatgccact gctcccacca cctccaccac ctgagccaca
     5701 gtcaagcgaa gaccccatca gcccacccga gcctcaggat ctgtcctcta gtatttgcaa
     5761 agaggaaaag atgcccagag caggcagcct ggagagtgat ggctgtccaa aagaacccgc
     5821 caagacccag cctatggtgg cagccgctgc aactaccacc acaaccacaa ctaccacagt
     5881 ggccgaaaaa tacaagcatc gcggcgaggg cgaacgaaag gacattgtgt caagctccat
     5941 gcccagacct aaccgggagg aaccagtcga tagtaggaca cccgtgactg agagagtctc
     6001 atagggcctg tcaggccaag cttccatcga tagacatgat aagatacatt gatgagtttg
     6061 gacaaaccac aacaagaatg cagtgaaaaa aatgctttat ttgtgaaatt tgtgatgcta
     6121 ttgctttatt tgtaaccatt ataagctgca ataaacaagt taacaacaac aattgcattc
     6181 attttatgtt tcaggttcag ggggagatgt gggaggtttt ttaaagcaag taaaacctct
     6241 acaaatgtgg taccgagggc ctatttccca tgattccttc atatttgcat atacgataca
     6301 aggctgttag agagataatt ggaattaatt tgactgtaaa cacaaagata ttagtacaaa
     6361 atacgtgacg tagaaagtaa taatttcttg ggtagtttgc agttttaaaa ttatgtttta
     6421 aaatggacta tcatatgctt accgtaactt gaaagtattt cgatttcttg gctttatata
     6481 tcttgtggaa aggacgaaac accggaagag cgagctcttc tgttttagag ctagaaatag
     6541 caagttaaaa taaggctagt ccgttatcaa cttgaaaaag tggcaccgag tcggtgcttt
     6601 tttgttttgg tacctaggcg agaccctgtc tcacaaaata aagtaagccc ggactgagtg
     6661 cggaaaggcg ggcctggcgg gtctggtctc cccatgcggg ccaccagagg ccctgcagcc
     6721 ttcagtcgct tgaaggggta atggcgcttc cactcacaaa catggcggac agagcgtgtg
     6781 aacgagatga acagcccctc aaaaatatgg ccgccgaggc tggacggccg tgccccagca
     6841 gcaccgcctc cgcgccccac gtgatctctc gccgggcaca gcgctgaccg cggaggtcca
     6901 accggaagaa tgtccggatt ggacattcgg aagagggccc gccttccctg gggaatctct
     6961 gcgcacgcgc agaacgcttc gaccaatgaa aacacaggaa gccgtccgcg caaccgcgtt
     7021 gcgtcacttc tgccgcccct gtttcaaggt atatagccgt agacggaact tcgcctttct
     7081 ctcggcctta gcgccatttt tttgggtgag tgttttttgg ttcctgcgtt gggattccgt
     7141 gtacaatcca tagacatctg acctcggcac ttagcatcat cacagcaaac taactgtagc
     7201 ctttctctct ttccctgtag aaacctctgc acctgaggcc accatgtcta gactggacaa
     7261 gagcaaagtc ataaacggcg ctctggaatt actcaatgga gtcggtatcg aaggcctgac
     7321 gacaaggaaa ctcgctcaaa agctgggagt tgagcagcct accctgtact ggcacgtgaa
     7381 gaacaagcgg gccctgctcg atgccctgcc aatcgagatg ctggacaggc atcataccca
     7441 cttctgcccc ctggaaggcg agtcatggca agactttctg cggaacaacg ccaagtcatt
     7501 ccgctgtgct ctcctctcac atcgcgacgg ggctaaagtg catctcggca cccgcccaac
     7561 agagaaacag tacgaaaccc tggaaaatca gctcgcgttc ctgtgtcagc aaggcttctc
     7621 cctggagaac gcactgtacg ctctgtccgc cgtgggccac tttacactgg gctgcgtatt
     7681 ggaggaacag gagcatcaag tagcaaaaga ggaaagagag acacctacca ccgattctat
     7741 gcccccactt ctgagacaag caattgagct gttcgaccgg cagggagccg aacctgcctt
     7801 ccttttcggc ctggaactaa tcatatgtgg cctggagaaa cagctaaagt gcgaaagcgg
     7861 cgggccggcc gacgcccttg acgattttga cttagacatg ctcccagccg atgcccttga
     7921 cgactttgac cttgatatgc tgcctgctga cgctcttgac gattttgacc ttgacatgct
     7981 ccccgggtcc ggcgctacta acttcagcct gctgaagcag gctggtgacg tcgaggagaa
     8041 tcctggtccc atgaaaaagc ctgaactcac cgcgacgtct gtcgagaagt ttctgatcga
     8101 aaagttcgac agcgtctccg acctgatgca gctctcggag ggcgaagaat ctcgtgcttt
     8161 cagcttcgat gtaggagggc gtggatatgt cctgcgggta aatagctgcg ccgatggttt
     8221 ctacaaagat cgttatgttt atcggcactt tgcatcggcc gcgctcccga ttccggaagt
     8281 gcttgacatt ggggaattca gcgagagcct gacctattgc atctcccgcc gtgcacaggg
     8341 tgtcatgttg caagacctgc ctgaaaccga actgcccgct gttctgcagc cggtcgcgga
     8401 ggccatggat gcgatcgctg cggccgatct tagccagacg agcgggttcg gcccattcgg
     8461 accgcaagga atcggtcaat acactacatg gcgtgatttc atatgcgcga ttgctgatcc
     8521 ccatgtgtat cactggcaaa ctgtgatgga cgacaccgtc agtgcgtccg tcgcgcaggc
     8581 tctcgatgag ctgatgcttt gggccgagga ctgccccgaa gtccggcacc tcgtgcacgc
     8641 ggatttcggc tccaacaatg tcctgacgga caatggccgc ataacagcgg tcattgactg
     8701 gagcgaggcg atgttcgggg attcccaata cgaggtcgcc aacatcttct tctggaggcc
     8761 gtggttggct tgtatggagc agcagacgcg ctacttcgag cggaggcatc cggagcttgc
     8821 aggatcgccg cggctccggg cgtatatgct ccgcattggt cttgaccaac tctatcagag
     8881 cttggttgac ggcaatttcg atgatgcagc ttgggcgcag ggtcgatgcg acgcaatcgt
     8941 ccgatccgga gccgggactg tcgggcgtac acaaatcgcc cgcagaagcg cggccgtctg
     9001 gaccgatggc tgtgtagaag tactcgccga tagtggaaac cgacgcccca gcactcgtcc
     9061 gagggcaaag gaatagcacc tggtgcatga cccgcaagcc cggtgcctga ttcgaaggcc
     9121 tgtcgtgaag cttggggatc aattctctag agctcgctga tcagcctcga ctgtgccttc
     9181 tagttgccag ccatctgttg tttgcccctc ccccgtgcct tccttgaccc tggaaggtgc
     9241 cactcccact gtcctttcct aataaaatga ggaaattgca tcgcattgtc tgagtaggtg
     9301 tcattctatt ctggggggtg gggtggggca ggacagcaag ggggaggatt gggaagacaa
     9361 tagcaggcat gctggggatg cggtgggctc tatggcttct gaggcggaaa gaaccagctg
     9421 ggcctactag ctactcggga ccccttaccg aaacatcgcc gcattctgca gaggagtcga
     9481 gtgtatgtaa acttctgacc cactgggaat gtgatgaaag aaataaaagc tgaaatgaat
     9541 cattctctct actattattc tgatatttca cattcttaaa ataaagtggt gatcctaact
     9601 gacctaagac agggaatttt tactaggatt aaatgtcagg aattgtgaaa aagtgagttt
     9661 aaatgtattt ggctaaggtg tatgtaaact tccgacttca actgtatagg gatccgcttc
     9721 ctcgctcact gactcgctgc gctcggtcgt tcggctgcgg cgagcggtat cagctcactc
     9781 aaaggcggta atacggttat ccacagaatc aggggataac gcaggaaaga acatgtgagc
     9841 aaaaggccag caaaaggcca ggaaccgtaa aaaggccgcg ttgctggcgt ttttccatag
     9901 gctccgcccc cctgacgagc atcacaaaaa tcgacgctca agtcagaggt ggcgaaaccc
     9961 gacaggacta taaagatacc aggcgtttcc ccctggaagc tccctcgtgc gctctcctgt
    10021 tccgaccctg ccgcttaccg gatacctgtc cgcctttctc ccttcgggaa gcgtggcgct
    10081 ttctcatagc tcacgctgta ggtatctcag ttcggtgtag gtcgttcgct ccaagctggg
    10141 ctgtgtgcac gaaccccccg ttcagcccga ccgctgcgcc ttatccggta actatcgtct
    10201 tgagtccaac ccggtaagac acgacttatc gccactggca gcagccactg gtaacaggat
    10261 tagcagagcg aggtatgtag gcggtgctac agagttcttg aagtggtggc ctaactacgg
    10321 ctacactaga agaacagtat ttggtatctg cgctctgctg aagccagtta ccttcggaaa
    10381 aagagttggt agctcttgat ccggcaaaca aaccaccgct ggtagcggtg gtttttttgt
    10441 ttgcaagcag cagattacgc gcagaaaaaa aggatctcaa gaagatcctt tgatcttttc
    10501 tacggggtct gacgctcagt ggaacgaaaa ctcacgttaa gggattttgg tcatgagatt
    10561 atcaaaaagg atcttcacct agatcctttt aaattaaaaa tgaagtttta aatcaatcta
    10621 aagtatatat gagtaaactt ggtctgacag ttaccaatgc ttaatcagtg aggcacctat
    10681 ctcagcgatc tgtctatttc gttcatccat agttgcctga ctccccgtcg tgtagataac
    10741 tacgatacgg gagggcttac catctggccc cagtgctgca atgataccgc gagacccacg
    10801 ctcaccggct ccagatttat cagcaataaa ccagccagcc ggaagggccg agcgcagaag
    10861 tggtcctgca actttatccg cctccatcca gtctattaat tgttgccggg aagctagagt
    10921 aagtagttcg ccagttaata gtttgcgcaa cgttgttgcc attgctacag gcatcgtggt
    10981 gtcacgctcg tcgtttggta tggcttcatt cagctccggt tcccaacgat caaggcgagt
    11041 tacatgatcc cccatgttgt gcaaaaaagc ggttagctcc ttcggtcctc cgatcgttgt
    11101 cagaagtaag ttggccgcag tgttatcact catggttatg gcagcactgc ataattctct
    11161 tactgtcatg ccatccgtaa gatgcttttc tgtgactggt gagtactcaa ccaagtcatt
    11221 ctgagaatag tgtatgcggc gaccgagttg ctcttgcccg gcgtcaatac gggataatac
    11281 cgcgccacat agcagaactt taaaagtgct catcattgga aaacgttctt cggggcgaaa
    11341 actctcaagg atcttaccgc tgttgagatc cagttcgatg taacccactc gtgcacccaa
    11401 ctgatcttca gcatctttta ctttcaccag cgtttctggg tgagcaaaaa caggaaggca
    11461 aaatgccgca aaaaagggaa taagggcgac acggaaatgt tgaatactca tactcttcct
    11521 ttttcaatat tattgaagca tttatcaggg ttattgtctc atgagcggat acatatttga
    11581 atgtatttag aaaaataaac aaataggggt tccgcgcaca tttccccgaa aagtgccacc
    11641 tgatgcggtg tgaaataccg cacagatgcg taaggagaaa ataccgcatc aggaaattgt
    11701 aagcgttaat attttgttaa aatt
//
