LOCUS       sequence-388993-       11264 bp    DNA     circular SYN 14-APR-2025
DEFINITION  (Empty Backbone) Inducible CRISPRi vector conferring puromycin 
            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 11264)
  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 11264)
  AUTHORS   .
  TITLE     Direct Submission
  JOURNAL   Exported Apr 14, 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-Pur-dCas9-KRAB-MeCP2_hU6-SapI
FEATURES             Location/Qualifiers
     source          1..11264
                     /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..8650
                     /codon_start=1
                     /gene="pac from Streptomyces alboniger"
                     /product="puromycin N-acetyltransferase"
                     /label=PuroR
                     /note="confers resistance to puromycin"
                     /translation="MTEYKPTVRLATRDDVPRAVRTLAAAFADYPATRHTVDPDRHIER
                     VTELQELFLTRVGLDIGKVWVADDGAAVAVWTTPESVEAGAVFAEIGPRMAELSGSRLA
                     AQQQMEGLLAPHRPKEPAWFLATVGVSPDHQGKGLGSAVVLPGVEAAERAGVPAFLETS
                     APRNLPFYERLGFTVTADVEVPEGPRTWCMTRKPGA"
     primer_bind     complement(8051..8070)
                     /label=Puro-R
                     /note="Puromycin resistance gene, reverse primer. Also 
                     called puro-variant-R"
     primer_bind     8547..8567
                     /label=Puro-F
                     /note="Puromycin resistance gene, forward primer"
     primer_bind     complement(8705..8722)
                     /label=BGH-rev
                     /note="Bovine growth hormone terminator, reverse primer. 
                     Also called BGH reverse"
     polyA_signal    8711..8935
                     /label=bGH poly(A) signal
                     /note="bovine growth hormone polyadenylation signal"
     primer_bind     complement(9261..9278)
                     /label=L4440
                     /note="L4440 vector, forward primer"
     rep_origin      complement(9432..10020)
                     /direction=LEFT
                     /label=ori
                     /note="high-copy-number ColE1/pMB1/pBR322/pUC origin of 
                     replication"
     primer_bind     complement(9512..9531)
                     /label=pBR322ori-F
                     /note="pBR322 origin, forward primer"
     CDS             complement(join(10191..10982,10983..11051))
                     /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     10814..10833
                     /label=Amp-R
                     /note="Ampicillin resistance gene, reverse primer"
     promoter        complement(11052..11156)
                     /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 atgaccgagt acaagcccac ggtgcgcctc gccacccgcg acgacgtccc
     8101 cagggccgta cgcaccctcg ccgccgcgtt cgccgactac cccgccacgc gccacaccgt
     8161 cgatccggac cgccacatcg agcgggtcac cgagctgcaa gaactcttcc tcacgcgcgt
     8221 cgggctcgac atcggcaagg tgtgggtcgc ggacgacggc gccgcggtgg cggtctggac
     8281 cacgccggag agcgtcgaag cgggggcggt gttcgccgag atcggcccgc gcatggccga
     8341 gttgagcggt tcccggctgg ccgcgcagca acagatggaa ggcctcctgg cgccgcaccg
     8401 gcccaaggag cccgcgtggt tcctggccac cgtcggcgtc tcgcccgacc accagggcaa
     8461 gggtctgggc agcgccgtcg tgctccccgg agtggaggcg gccgagcgcg ccggggtgcc
     8521 cgccttcctg gagacctccg cgccccgcaa cctccccttc tacgagcggc tcggcttcac
     8581 cgtcaccgcc gacgtcgagg tgcccgaagg accgcgcacc tggtgcatga cccgcaagcc
     8641 cggtgcctga ttcgaaggcc tgtcgtgaag cttggggatc aattctctag agctcgctga
     8701 tcagcctcga ctgtgccttc tagttgccag ccatctgttg tttgcccctc ccccgtgcct
     8761 tccttgaccc tggaaggtgc cactcccact gtcctttcct aataaaatga ggaaattgca
     8821 tcgcattgtc tgagtaggtg tcattctatt ctggggggtg gggtggggca ggacagcaag
     8881 ggggaggatt gggaagacaa tagcaggcat gctggggatg cggtgggctc tatggcttct
     8941 gaggcggaaa gaaccagctg ggcctactag ctactcggga ccccttaccg aaacatcgcc
     9001 gcattctgca gaggagtcga gtgtatgtaa acttctgacc cactgggaat gtgatgaaag
     9061 aaataaaagc tgaaatgaat cattctctct actattattc tgatatttca cattcttaaa
     9121 ataaagtggt gatcctaact gacctaagac agggaatttt tactaggatt aaatgtcagg
     9181 aattgtgaaa aagtgagttt aaatgtattt ggctaaggtg tatgtaaact tccgacttca
     9241 actgtatagg gatccgcttc ctcgctcact gactcgctgc gctcggtcgt tcggctgcgg
     9301 cgagcggtat cagctcactc aaaggcggta atacggttat ccacagaatc aggggataac
     9361 gcaggaaaga acatgtgagc aaaaggccag caaaaggcca ggaaccgtaa aaaggccgcg
     9421 ttgctggcgt ttttccatag gctccgcccc cctgacgagc atcacaaaaa tcgacgctca
     9481 agtcagaggt ggcgaaaccc gacaggacta taaagatacc aggcgtttcc ccctggaagc
     9541 tccctcgtgc gctctcctgt tccgaccctg ccgcttaccg gatacctgtc cgcctttctc
     9601 ccttcgggaa gcgtggcgct ttctcatagc tcacgctgta ggtatctcag ttcggtgtag
     9661 gtcgttcgct ccaagctggg ctgtgtgcac gaaccccccg ttcagcccga ccgctgcgcc
     9721 ttatccggta actatcgtct tgagtccaac ccggtaagac acgacttatc gccactggca
     9781 gcagccactg gtaacaggat tagcagagcg aggtatgtag gcggtgctac agagttcttg
     9841 aagtggtggc ctaactacgg ctacactaga agaacagtat ttggtatctg cgctctgctg
     9901 aagccagtta ccttcggaaa aagagttggt agctcttgat ccggcaaaca aaccaccgct
     9961 ggtagcggtg gtttttttgt ttgcaagcag cagattacgc gcagaaaaaa aggatctcaa
    10021 gaagatcctt tgatcttttc tacggggtct gacgctcagt ggaacgaaaa ctcacgttaa
    10081 gggattttgg tcatgagatt atcaaaaagg atcttcacct agatcctttt aaattaaaaa
    10141 tgaagtttta aatcaatcta aagtatatat gagtaaactt ggtctgacag ttaccaatgc
    10201 ttaatcagtg aggcacctat ctcagcgatc tgtctatttc gttcatccat agttgcctga
    10261 ctccccgtcg tgtagataac tacgatacgg gagggcttac catctggccc cagtgctgca
    10321 atgataccgc gagacccacg ctcaccggct ccagatttat cagcaataaa ccagccagcc
    10381 ggaagggccg agcgcagaag tggtcctgca actttatccg cctccatcca gtctattaat
    10441 tgttgccggg aagctagagt aagtagttcg ccagttaata gtttgcgcaa cgttgttgcc
    10501 attgctacag gcatcgtggt gtcacgctcg tcgtttggta tggcttcatt cagctccggt
    10561 tcccaacgat caaggcgagt tacatgatcc cccatgttgt gcaaaaaagc ggttagctcc
    10621 ttcggtcctc cgatcgttgt cagaagtaag ttggccgcag tgttatcact catggttatg
    10681 gcagcactgc ataattctct tactgtcatg ccatccgtaa gatgcttttc tgtgactggt
    10741 gagtactcaa ccaagtcatt ctgagaatag tgtatgcggc gaccgagttg ctcttgcccg
    10801 gcgtcaatac gggataatac cgcgccacat agcagaactt taaaagtgct catcattgga
    10861 aaacgttctt cggggcgaaa actctcaagg atcttaccgc tgttgagatc cagttcgatg
    10921 taacccactc gtgcacccaa ctgatcttca gcatctttta ctttcaccag cgtttctggg
    10981 tgagcaaaaa caggaaggca aaatgccgca aaaaagggaa taagggcgac acggaaatgt
    11041 tgaatactca tactcttcct ttttcaatat tattgaagca tttatcaggg ttattgtctc
    11101 atgagcggat acatatttga atgtatttag aaaaataaac aaataggggt tccgcgcaca
    11161 tttccccgaa aagtgccacc tgatgcggtg tgaaataccg cacagatgcg taaggagaaa
    11221 ataccgcatc aggaaattgt aagcgttaat attttgttaa aatt
//
