LOCUS       sequence-401799-       13114 bp    DNA     circular SYN 15-DEC-2025
DEFINITION  Lentiviral expression of S. pyogenes dead Cas9 
            (dCas9/dSpCas9/SpdCas9) fused to the KRAB domain as well as a gRNA 
            targeting the tight TRE promoter and the blasticidin resistance 
            gene..
ACCESSION   .
VERSION     .
KEYWORDS    .
SOURCE      synthetic DNA construct
  ORGANISM  synthetic DNA construct
REFERENCE   1  (bases 1 to 13114)
  TITLE     CRISPRi plasmids for reduced leakiness of doxycycline-inducible 
            tight TRE promoter
  JOURNAL   Unpublished
REFERENCE   2  (bases 1 to 13114)
  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: 
            "Unpublished"
COMMENT     Sequence Label: pLenti-dCas9-KRAB-gRNA-TRE-blast
FEATURES             Location/Qualifiers
     source          1..13114
                     /mol_type="other DNA"
                     /organism="synthetic DNA construct"
     misc_RNA        1..76
                     /label=gRNA scaffold
                     /note="guide RNA scaffold for the Streptococcus pyogenes 
                     CRISPR/Cas9 system"
     promoter        138..349
                     /label=EF-1-alpha core promoter
                     /note="core promoter for human elongation factor 
                     EF-1-alpha"
     regulatory      362..371
                     /label=Kozak sequence
                     /note="vertebrate consensus sequence for strong initiation 
                     of translation (Kozak, 1987)"
                     /regulatory_class="other"
     CDS             398..583
                     /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
                     QLTKPDVILRLEKGEEP"
     CDS             605..4708
                     /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="MDKKYSIGLAIGTNSVGWAVITDEYKVPSKKFKVLGNTDRHSIKK
                     NLIGALLFDSGETAEATRLKRTARRRYTRRKNRICYLQEIFSNEMAKVDDSFFHRLEES
                     FLVEEDKKHERHPIFGNIVDEVAYHEKYPTIYHLRKKLVDSTDKADLRLIYLALAHMIK
                     FRGHFLIEGDLNPDNSDVDKLFIQLVQTYNQLFEENPINASGVDAKAILSARLSKSRRL
                     ENLIAQLPGEKKNGLFGNLIALSLGLTPNFKSNFDLAEDAKLQLSKDTYDDDLDNLLAQ
                     IGDQYADLFLAAKNLSDAILLSDILRVNTEITKAPLSASMIKRYDEHHQDLTLLKALVR
                     QQLPEKYKEIFFDQSKNGYAGYIDGGASQEEFYKFIKPILEKMDGTEELLVKLNREDLL
                     RKQRTFDNGSIPHQIHLGELHAILRRQEDFYPFLKDNREKIEKILTFRIPYYVGPLARG
                     NSRFAWMTRKSEETITPWNFEEVVDKGASAQSFIERMTNFDKNLPNEKVLPKHSLLYEY
                     FTVYNELTKVKYVTEGMRKPAFLSGEQKKAIVDLLFKTNRKVTVKQLKEDYFKKIECFD
                     SVEISGVEDRFNASLGTYHDLLKIIKDKDFLDNEENEDILEDIVLTLTLFEDREMIEER
                     LKTYAHLFDDKVMKQLKRRRYTGWGRLSRKLINGIRDKQSGKTILDFLKSDGFANRNFM
                     QLIHDDSLTFKEDIQKAQVSGQGDSLHEHIANLAGSPAIKKGILQTVKVVDELVKVMGR
                     HKPENIVIEMARENQTTQKGQKNSRERMKRIEEGIKELGSQILKEHPVENTQLQNEKLY
                     LYYLQNGRDMYVDQELDINRLSDYDVDAIVPQSFLKDDSIDNKVLTRSDKNRGKSDNVP
                     SEEVVKKMKNYWRQLLNAKLITQRKFDNLTKAERGGLSELDKAGFIKRQLVETRQITKH
                     VAQILDSRMNTKYDENDKLIREVKVITLKSKLVSDFRKDFQFYKVREINNYHHAHDAYL
                     NAVVGTALIKKYPKLESEFVYGDYKVYDVRKMIAKSEQEIGKATAKYFFYSNIMNFFKT
                     EITLANGEIRKRPLIETNGETGEIVWDKGRDFATVRKVLSMPQVNIVKKTEVQTGGFSK
                     ESILPKRNSDKLIARKKDWDPKKYGGFDSPTVAYSVLVVAKVEKGKSKKLKSVKELLGI
                     TIMERSSFEKNPIDFLEAKGYKEVKKDLIIKLPKYSLFELENGRKRMLASAGELQKGNE
                     LALPSKYVNFLYLASHYEKLKGSPEDNEQKQLFVEQHKHYLDEIIEQISEFSKRVILAD
                     ANLDKVLSAYNKHRDKPIREQAENIIHLFTLTSLGAPAAFKYFDTTIDRKRYTSTKEVL
                     DATLIHQSITGLYETRIDLSQLGGD"
     CDS             4712..4738
                     /codon_start=1
                     /product="HA (human influenza hemagglutinin) epitope tag"
                     /label=HA
                     /translation="YPYDVPDYA"
     CDS             4718..4744
                     /codon_start=1
                     /product="HA (human influenza hemagglutinin) epitope tag"
                     /label=HA
                     /note="This is a variant of the standard HA tag, which has 
                     sequence YPYDVPDYA. Some anti-HA antibodies may not 
                     recognize this variant."
                     /translation="YDVPDYASL"
     CDS             4757..4777
                     /codon_start=1
                     /product="nuclear localization signal of SV40 (simian virus
                     40) large T antigen"
                     /label=SV40 NLS
                     /translation="PKKKRKV"
     CDS             4784..4804
                     /codon_start=1
                     /product="nuclear localization signal of SV40 (simian virus
                     40) large T antigen"
                     /label=SV40 NLS
                     /translation="PKKKRKV"
     CDS             4850..4906
                     /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             4907..5305
                     /codon_start=1
                     /gene="Aspergillus terreus BSD"
                     /product="blasticidin S deaminase"
                     /label=BSD
                     /note="confers resistance to blasticidin"
                     /translation="MAKPLSQEESTLIERATATINSIPISEDYSVASAALSSDGRIFTG
                     VNVYHFTGGPCAELVVLGTAAAAAAGNLTCIVAIGNENRGILSPCGRCRQVLLDLHPGI
                     KAIVKDSDGQPTAVGIRELLPSGYVWEG"
     misc_feature    5330..5918
                     /label=WPRE
                     /note="woodchuck hepatitis virus posttranscriptional 
                     regulatory element"
     primer_bind     complement(5383..5403)
                     /label=WPRE-R
                     /note="WPRE, reverse primer"
     CDS             complement(5801..5812)
                     /codon_start=1
                     /product="Factor Xa recognition and cleavage site"
                     /label=Factor Xa site
                     /translation="IEGR"
     LTR             5990..6223
                     /label=3' LTR (Delta-U3)
                     /note="self-inactivating 3' long terminal repeat (LTR) from
                     HIV-1"
     primer_bind     complement(6249..6266)
                     /label=BGH-rev
                     /note="Bovine growth hormone terminator, reverse primer. 
                     Also called BGH reverse"
     polyA_signal    6255..6479
                     /label=bGH poly(A) signal
                     /note="bovine growth hormone polyadenylation signal"
     rep_origin      6525..6953
                     /direction=RIGHT
                     /label=f1 ori
                     /note="f1 bacteriophage origin of replication; arrow 
                     indicates direction of (+) strand synthesis"
     primer_bind     complement(6612..6631)
                     /label=F1ori-R
                     /note="F1 origin, reverse primer"
     primer_bind     6822..6843
                     /label=F1ori-F
                     /note="F1 origin, forward primer"
     primer_bind     complement(6962..6982)
                     /label=pBABE 3'
                     /note="SV40 enhancer, reverse primer for pBABE vectors"
     promoter        6967..7296
                     /label=SV40 promoter
                     /note="SV40 enhancer and early promoter"
     rep_origin      7147..7282
                     /label=SV40 ori
                     /note="SV40 origin of replication"
     primer_bind     7209..7228
                     /label=SV40pro-F
                     /note="SV40 promoter/origin, forward primer"
     promoter        7344..7391
                     /label=EM7 promoter
                     /note="synthetic bacterial promoter "
     CDS             7410..7784
                     /codon_start=1
                     /gene="Sh ble from Streptoalloteichus hindustanus"
                     /product="antibiotic-binding protein"
                     /label=BleoR
                     /note="confers resistance to bleomycin, phleomycin, and 
                     Zeocin(TM)"
                     /translation="MAKLTSAVPVLTARDVAGAVEFWTDRLGFSRDFVEDDFAGVVRDD
                     VTLFISAVQDQVVPDNTLAWVWVRGLDELYAEWSEVVSTNFRDASGPAMTEIGEQPWGR
                     EFALRDPAGNCVHFVAEEQD"
     polyA_signal    7914..8035
                     /label=SV40 poly(A) signal
                     /note="SV40 polyadenylation signal"
     primer_bind     complement(7951..7970)
                     /label=SV40pA-R
                     /note="SV40 polyA, reverse primer"
     primer_bind     8005..8024
                     /label=EBV-rev
                     /note="SV40 polyA terminator, reverse primer"
     primer_bind     complement(8084..8100)
                     /label=M13 rev
                     /note="common sequencing primer, one of multiple similar 
                     variants"
     primer_bind     complement(8084..8100)
                     /label=M13 Reverse
                     /note="In lacZ gene. Also called M13-rev"
     primer_bind     complement(8097..8119)
                     /label=M13/pUC Reverse
                     /note="In lacZ gene"
     protein_bind    8108..8124
                     /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(8132..8138,8139..8156,8157..8162))
                     /label=lac promoter
                     /note="promoter for the E. coli lac operon"
     protein_bind    8177..8198
                     /label=CAP binding site
                     /bound_moiety="E. coli catabolite activator protein"
                     /note="CAP binding activates transcription in the presence 
                     of cAMP."
     primer_bind     complement(8315..8332)
                     /label=L4440
                     /note="L4440 vector, forward primer"
     rep_origin      complement(8486..9074)
                     /direction=LEFT
                     /label=ori
                     /note="high-copy-number ColE1/pMB1/pBR322/pUC origin of 
                     replication"
     primer_bind     complement(8566..8585)
                     /label=pBR322ori-F
                     /note="pBR322 origin, forward primer"
     CDS             complement(join(9245..10036,10037..10105))
                     /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     9868..9887
                     /label=Amp-R
                     /note="Ampicillin resistance gene, reverse primer"
     promoter        complement(10106..10210)
                     /gene="bla"
                     /label=AmpR promoter
     primer_bind     complement(10285..10304)
                     /label=pRS-marker
                     /note="pRS vectors, use to sequence yeast selectable 
                     marker"
     enhancer        10476..10855
                     /label=CMV enhancer
                     /note="human cytomegalovirus immediate early enhancer"
     promoter        10857..11055
                     /label=CMV promoter
                     /note="human cytomegalovirus (CMV) immediate early 
                     promoter"
     primer_bind     11010..11030
                     /label=CMV-F
                     /note="Human CMV immediate early promoter, forward primer"
     LTR             11073..11253
                     /label=5' LTR (truncated)
                     /note="truncated 5' long terminal repeat (LTR) from HIV-1"
     misc_feature    11300..11425
                     /label=HIV-1 Psi
                     /note="packaging signal of human immunodeficiency virus 
                     type 1"
     misc_feature    11918..12151
                     /label=RRE
                     /note="The Rev response element (RRE) of HIV-1 allows for 
                     Rev-dependent mRNA export from the nucleus to the 
                     cytoplasm."
     CDS             12336..12380
                     /codon_start=1
                     /product="antigenic peptide corresponding to amino acids 
                     655 to 669 of the HIV envelope protein gp41 (Lutje Hulsik 
                     et al., 2013)"
                     /label=gp41 peptide
                     /note="recognized by the 2H10 single-chain llama nanobody"
                     /translation="KNEQELLELDKWASL"
     misc_feature    12678..12795
                     /label=cPPT/CTS
                     /note="central polypurine tract and central termination 
                     sequence of HIV-1"
     promoter        12846..13094
                     /label=U6 promoter
                     /note="RNA polymerase III promoter for human U6 snRNA 
                     (Domitrovich & Kunkel, 2003)"
     primer_bind     12846..12866
                     /label=hU6-F
                     /note="Human U6 promoter, forward primer"
     primer_bind     13017..13036
                     /label=LKO.1 5'
                     /note="Human U6 promoter, forward primer"
ORIGIN
        1 gttttagagc tagaaatagc aagttaaaat aaggctagtc cgttatcaac ttgaaaaagt
       61 ggcaccgagt cggtgctttt ttgaattcgc tagctaggtc ttgaaaggag tgggaattgg
      121 ctccggtgcc cgtcagtggg cagagcgcac atcgcccaca gtccccgaga agttgggggg
      181 aggggtcggc aattgatccg gtgcctagag aaggtggcgc ggggtaaact gggaaagtga
      241 tgtcgtgtac tggctccgcc tttttcccga gggtggggga gaaccgtata taagtgcagt
      301 agtcgccgtg aacgttcttt ttcgcaacgg gtttgccgcc agaacacagg accggtgatc
      361 cgccaccatg gatgctaagt cactaactgc ctggtcccgg acactggtga ccttcaagga
      421 tgtatttgtg gacttcacca gggaggagtg gaagctgctg gacactgctc agcagatcgt
      481 gtacagaaat gtgatgctgg agaactataa gaacctggtt tccttgggtt atcagcttac
      541 taagccagat gtgatcctcc ggttggagaa gggagaagag cccggcggtt ccggcggagg
      601 gtcgatggac aagaagtaca gcatcggcct ggccatcggc accaactctg tgggctgggc
      661 cgtgatcacc gacgagtaca aggtgcccag caagaaattc aaggtgctgg gcaacaccga
      721 ccggcacagc atcaagaaga acctgatcgg cgccctgctg ttcgacagcg gagaaacagc
      781 cgaggccacc cggctgaaga gaaccgccag aagaagatac accagacgga agaaccggat
      841 ctgctatctg caagagatct tcagcaacga gatggccaag gtggacgaca gcttcttcca
      901 cagactggaa gagtccttcc tggtggaaga ggataagaag cacgagcggc accccatctt
      961 cggcaacatc gtggacgagg tggcctacca cgagaagtac cccaccatct accacctgag
     1021 aaagaaactg gtggacagca ccgacaaggc cgacctgcgg ctgatctatc tggccctggc
     1081 ccacatgatc aagttccggg gccacttcct gatcgagggc gacctgaacc ccgacaacag
     1141 cgacgtggac aagctgttca tccagctggt gcagacctac aaccagctgt tcgaggaaaa
     1201 ccccatcaac gccagcggcg tggacgccaa ggccatcctg tctgccagac tgagcaagag
     1261 cagacggctg gaaaatctga tcgcccagct gcccggcgag aagaagaatg gcctgttcgg
     1321 caacctgatt gccctgagcc tgggcctgac ccccaacttc aagagcaact tcgacctggc
     1381 cgaggatgcc aaactgcagc tgagcaagga cacctacgac gacgacctgg acaacctgct
     1441 ggcccagatc ggcgaccagt acgccgacct gtttctggcc gccaagaacc tgtccgacgc
     1501 catcctgctg agcgacatcc tgagagtgaa caccgagatc accaaggccc ccctgagcgc
     1561 ctctatgatc aagagatacg acgagcacca ccaggacctg accctgctga aagctctcgt
     1621 gcggcagcag ctgcctgaga agtacaaaga gattttcttc gaccagagca agaacggcta
     1681 cgccggctac atcgatggcg gagccagcca ggaagagttc tacaagttca tcaagcccat
     1741 cctggaaaag atggacggca ccgaggaact gctcgtgaag ctgaacagag aggacctgct
     1801 gcggaagcag cggaccttcg acaacggcag catcccccac cagatccacc tgggagagct
     1861 gcacgccatt ctgcggcggc aggaagattt ttacccattc ctgaaggaca accgggaaaa
     1921 gatcgagaag atcctgacct tccgcatccc ctactacgtg ggccctctgg ccaggggaaa
     1981 cagcagattc gcctggatga ccagaaagag cgaggaaacc atcaccccct ggaacttcga
     2041 ggaagtggtg gacaagggcg ccagcgccca gagcttcatc gagcggatga ccaacttcga
     2101 taagaacctg cccaacgaga aggtgctgcc caagcacagc ctgctgtacg agtacttcac
     2161 cgtgtacaac gagctgacca aagtgaaata cgtgaccgag ggaatgagaa agcccgcctt
     2221 cctgagcggc gagcagaaaa aagccatcgt ggacctgctg ttcaagacca accggaaagt
     2281 gaccgtgaag cagctgaaag aggactactt caagaaaatc gagtgcttcg actccgtgga
     2341 aatctccggc gtggaagatc ggttcaacgc ctccctgggc acataccacg atctgctgaa
     2401 aattatcaag gacaaggact tcctggacaa tgaggaaaac gaggacattc tggaagatat
     2461 cgtgctgacc ctgacactgt ttgaggacag agagatgatc gaggaacggc tgaaaaccta
     2521 tgcccacctg ttcgacgaca aagtgatgaa gcagctgaag cggcggagat acaccggctg
     2581 gggcaggctg agccggaagc tgatcaacgg catccgggac aagcagtccg gcaagacaat
     2641 cctggatttc ctgaagtccg acggcttcgc caacagaaac ttcatgcagc tgatccacga
     2701 cgacagcctg acctttaaag aggacatcca gaaagcccag gtgtccggcc agggcgatag
     2761 cctgcacgag cacattgcca atctggccgg cagccccgcc attaagaagg gcatcctgca
     2821 gacagtgaag gtggtggacg agctcgtgaa agtgatgggc cggcacaagc ccgagaacat
     2881 cgtgatcgaa atggccagag agaaccagac cacccagaag ggacagaaga acagccgcga
     2941 gagaatgaag cggatcgaag agggcatcaa agagctgggc agccagatcc tgaaagaaca
     3001 ccccgtggaa aacacccagc tgcagaacga gaagctgtac ctgtactacc tgcagaatgg
     3061 gcgggatatg tacgtggacc aggaactgga catcaaccgg ctgtccgact acgatgtgga
     3121 cgctatcgtg cctcagagct ttctgaagga cgactccatc gataacaaag tgctgactcg
     3181 gagcgacaag aaccggggca agagcgacaa cgtgccctcc gaagaggtcg tgaagaagat
     3241 gaagaactac tggcgccagc tgctgaatgc caagctgatt acccagagga agttcgacaa
     3301 tctgaccaag gccgagagag gcggcctgag cgaactggat aaggccggct tcatcaagag
     3361 acagctggtg gaaacccggc agatcacaaa gcacgtggca cagatcctgg actcccggat
     3421 gaacactaag tacgacgaga acgacaaact gatccgggaa gtgaaagtga tcaccctgaa
     3481 gtccaagctg gtgtccgatt tccggaagga tttccagttt tacaaagtgc gcgagatcaa
     3541 caactaccac cacgcccacg acgcctacct gaacgccgtc gtgggaaccg ccctgatcaa
     3601 aaagtaccct aagctggaaa gcgagttcgt gtacggcgac tacaaggtgt acgacgtgcg
     3661 gaagatgatc gccaagagcg agcaggaaat cggcaaggct accgccaagt acttcttcta
     3721 cagcaacatc atgaactttt tcaagaccga gattaccctg gccaacggcg agatccggaa
     3781 gcggcctctg atcgagacaa acggcgaaac aggcgagatc gtgtgggata agggccggga
     3841 ctttgccacc gtgcggaaag tgctgtctat gccccaagtg aatatcgtga aaaagaccga
     3901 ggtgcagaca ggcggcttca gcaaagagtc tatcctgccc aagaggaaca gcgacaagct
     3961 gatcgccaga aagaaggact gggaccctaa gaagtacggc ggcttcgaca gccccaccgt
     4021 ggcctattct gtgctggtgg tggccaaagt ggaaaagggc aagtccaaga aactgaagag
     4081 tgtgaaagag ctgctgggga tcaccatcat ggaaagaagc agcttcgaga agaatcccat
     4141 cgactttctg gaagccaagg gctacaaaga agtgaaaaag gacctgatca tcaagctgcc
     4201 taagtactcc ctgttcgagc tggaaaacgg ccggaagaga atgctggcct ctgccggcga
     4261 actgcagaag ggaaacgaac tggccctgcc ctccaaatat gtgaacttcc tgtacctggc
     4321 cagccactat gagaagctga agggctcccc cgaggataat gagcagaaac agctgtttgt
     4381 ggaacagcac aaacactacc tggacgagat catcgagcag atcagcgagt tctccaagag
     4441 agtgatcctg gccgacgcta atctggacaa ggtgctgagc gcctacaaca agcacagaga
     4501 caagcctatc agagagcagg ccgagaatat catccacctg tttaccctga ccagtctggg
     4561 agcccctgcc gccttcaagt actttgacac caccatcgac cggaagaggt acaccagcac
     4621 caaagaggtg ctggacgcca ccctgatcca ccagagcatc accggcctgt acgagacacg
     4681 gatcgacctg tctcagctgg gaggcgacgc ctatccctat gacgtgcccg attatgccag
     4741 cctgggcagc ggctccccca agaaaaaacg caaggtggaa gatcctaaga aaaagcggaa
     4801 agtggacggc attggtagtg ggagcaacgg cagcagcggg gatccggccg caacaaactt
     4861 ctctctgctg aaacaagccg gagatgtcga agagaatcct ggaccgatgg ccaagccttt
     4921 gtctcaagaa gaatccaccc tcattgaaag agcaacggct acaatcaaca gcatccccat
     4981 ctctgaagac tacagcgtcg ccagcgcagc tctctctagc gacggccgca tcttcactgg
     5041 tgtcaatgta tatcatttta ctgggggacc ttgtgcagaa ctcgtggtgc tgggcactgc
     5101 tgctgctgcg gcagctggca acctgacttg tatcgtcgcg atcggaaatg agaacagggg
     5161 catcttgagc ccctgcggac ggtgccgaca ggtgcttctc gatctgcatc ctgggatcaa
     5221 agccatagtg aaggacagtg atggacagcc gacggcagtt gggattcgtg aattgctgcc
     5281 ctctggttat gtgtgggagg gctaagaatt cgatatcaag cttatcggta atcaacctct
     5341 ggattacaaa atttgtgaaa gattgactgg tattcttaac tatgttgctc cttttacgct
     5401 atgtggatac gctgctttaa tgcctttgta tcatgctatt gcttcccgta tggctttcat
     5461 tttctcctcc ttgtataaat cctggttgct gtctctttat gaggagttgt ggcccgttgt
     5521 caggcaacgt ggcgtggtgt gcactgtgtt tgctgacgca acccccactg gttggggcat
     5581 tgccaccacc tgtcagctcc tttccgggac tttcgctttc cccctcccta ttgccacggc
     5641 ggaactcatc gccgcctgcc ttgcccgctg ctggacaggg gctcggctgt tgggcactga
     5701 caattccgtg gtgttgtcgg ggaaatcatc gtcctttcct tggctgctcg cctgtgttgc
     5761 cacctggatt ctgcgcggga cgtccttctg ctacgtccct tcggccctca atccagcgga
     5821 ccttccttcc cgcggcctgc tgccggctct gcggcctctt ccgcgtcttc gccttcgccc
     5881 tcagacgagt cggatctccc tttgggccgc ctccccgcgt cgactttaag accaatgact
     5941 tacaaggcag ctgtagatct tagccacttt ttaaaagaaa aggggggact ggaagggcta
     6001 attcactccc aacgaagaca agatctgctt tttgcttgta ctgggtctct ctggttagac
     6061 cagatctgag cctgggagct ctctggctaa ctagggaacc cactgcttaa gcctcaataa
     6121 agcttgcctt gagtgcttca agtagtgtgt gcccgtctgt tgtgtgactc tggtaactag
     6181 agatccctca gaccctttta gtcagtgtgg aaaatctcta gcagggcccg tttaaacccg
     6241 ctgatcagcc tcgactgtgc cttctagttg ccagccatct gttgtttgcc cctcccccgt
     6301 gccttccttg accctggaag gtgccactcc cactgtcctt tcctaataaa atgaggaaat
     6361 tgcatcgcat tgtctgagta ggtgtcattc tattctgggg ggtggggtgg ggcaggacag
     6421 caagggggag gattgggaag acaatagcag gcatgctggg gatgcggtgg gctctatggc
     6481 ttctgaggcg gaaagaacca gctggggctc tagggggtat ccccacgcgc cctgtagcgg
     6541 cgcattaagc gcggcgggtg tggtggttac gcgcagcgtg accgctacac ttgccagcgc
     6601 cctagcgccc gctcctttcg ctttcttccc ttcctttctc gccacgttcg ccggctttcc
     6661 ccgtcaagct ctaaatcggg ggctcccttt agggttccga tttagtgctt tacggcacct
     6721 cgaccccaaa aaacttgatt agggtgatgg ttcacgtagt gggccatcgc cctgatagac
     6781 ggtttttcgc cctttgacgt tggagtccac gttctttaat agtggactct tgttccaaac
     6841 tggaacaaca ctcaacccta tctcggtcta ttcttttgat ttataaggga ttttgccgat
     6901 ttcggcctat tggttaaaaa atgagctgat ttaacaaaaa tttaacgcga attaattctg
     6961 tggaatgtgt gtcagttagg gtgtggaaag tccccaggct ccccagcagg cagaagtatg
     7021 caaagcatgc atctcaatta gtcagcaacc aggtgtggaa agtccccagg ctccccagca
     7081 ggcagaagta tgcaaagcat gcatctcaat tagtcagcaa ccatagtccc gcccctaact
     7141 ccgcccatcc cgcccctaac tccgcccagt tccgcccatt ctccgcccca tggctgacta
     7201 atttttttta tttatgcaga ggccgaggcc gcctctgcct ctgagctatt ccagaagtag
     7261 tgaggaggct tttttggagg cctaggcttt tgcaaaaagc tcccgggagc ttgtatatcc
     7321 attttcggat ctgatcagca cgtgttgaca attaatcatc ggcatagtat atcggcatag
     7381 tataatacga caaggtgagg aactaaacca tggccaagtt gaccagtgcc gttccggtgc
     7441 tcaccgcgcg cgacgtcgcc ggagcggtcg agttctggac cgaccggctc gggttctccc
     7501 gggacttcgt ggaggacgac ttcgccggtg tggtccggga cgacgtgacc ctgttcatca
     7561 gcgcggtcca ggaccaggtg gtgccggaca acaccctggc ctgggtgtgg gtgcgcggcc
     7621 tggacgagct gtacgccgag tggtcggagg tcgtgtccac gaacttccgg gacgcctccg
     7681 ggccggccat gaccgagatc ggcgagcagc cgtgggggcg ggagttcgcc ctgcgcgacc
     7741 cggccggcaa ctgcgtgcac ttcgtggccg aggagcagga ctgacacgtg ctacgagatt
     7801 tcgattccac cgccgccttc tatgaaaggt tgggcttcgg aatcgttttc cgggacgccg
     7861 gctggatgat cctccagcgc ggggatctca tgctggagtt cttcgcccac cccaacttgt
     7921 ttattgcagc ttataatggt tacaaataaa gcaatagcat cacaaatttc acaaataaag
     7981 catttttttc actgcattct agttgtggtt tgtccaaact catcaatgta tcttatcatg
     8041 tctgtatacc gtcgacctct agctagagct tggcgtaatc atggtcatag ctgtttcctg
     8101 tgtgaaattg ttatccgctc acaattccac acaacatacg agccggaagc ataaagtgta
     8161 aagcctgggg tgcctaatga gtgagctaac tcacattaat tgcgttgcgc tcactgcccg
     8221 ctttccagtc gggaaacctg tcgtgccagc tgcattaatg aatcggccaa cgcgcgggga
     8281 gaggcggttt gcgtattggg cgctcttccg cttcctcgct cactgactcg ctgcgctcgg
     8341 tcgttcggct gcggcgagcg gtatcagctc actcaaaggc ggtaatacgg ttatccacag
     8401 aatcagggga taacgcagga aagaacatgt gagcaaaagg ccagcaaaag gccaggaacc
     8461 gtaaaaaggc cgcgttgctg gcgtttttcc ataggctccg cccccctgac gagcatcaca
     8521 aaaatcgacg ctcaagtcag aggtggcgaa acccgacagg actataaaga taccaggcgt
     8581 ttccccctgg aagctccctc gtgcgctctc ctgttccgac cctgccgctt accggatacc
     8641 tgtccgcctt tctcccttcg ggaagcgtgg cgctttctca tagctcacgc tgtaggtatc
     8701 tcagttcggt gtaggtcgtt cgctccaagc tgggctgtgt gcacgaaccc cccgttcagc
     8761 ccgaccgctg cgccttatcc ggtaactatc gtcttgagtc caacccggta agacacgact
     8821 tatcgccact ggcagcagcc actggtaaca ggattagcag agcgaggtat gtaggcggtg
     8881 ctacagagtt cttgaagtgg tggcctaact acggctacac tagaagaaca gtatttggta
     8941 tctgcgctct gctgaagcca gttaccttcg gaaaaagagt tggtagctct tgatccggca
     9001 aacaaaccac cgctggtagc ggtggttttt ttgtttgcaa gcagcagatt acgcgcagaa
     9061 aaaaaggatc tcaagaagat cctttgatct tttctacggg gtctgacgct cagtggaacg
     9121 aaaactcacg ttaagggatt ttggtcatga gattatcaaa aaggatcttc acctagatcc
     9181 ttttaaatta aaaatgaagt tttaaatcaa tctaaagtat atatgagtaa acttggtctg
     9241 acagttacca atgcttaatc agtgaggcac ctatctcagc gatctgtcta tttcgttcat
     9301 ccatagttgc ctgactcccc gtcgtgtaga taactacgat acgggagggc ttaccatctg
     9361 gccccagtgc tgcaatgata ccgcgagacc cacgctcacc ggctccagat ttatcagcaa
     9421 taaaccagcc agccggaagg gccgagcgca gaagtggtcc tgcaacttta tccgcctcca
     9481 tccagtctat taattgttgc cgggaagcta gagtaagtag ttcgccagtt aatagtttgc
     9541 gcaacgttgt tgccattgct acaggcatcg tggtgtcacg ctcgtcgttt ggtatggctt
     9601 cattcagctc cggttcccaa cgatcaaggc gagttacatg atcccccatg ttgtgcaaaa
     9661 aagcggttag ctccttcggt cctccgatcg ttgtcagaag taagttggcc gcagtgttat
     9721 cactcatggt tatggcagca ctgcataatt ctcttactgt catgccatcc gtaagatgct
     9781 tttctgtgac tggtgagtac tcaaccaagt cattctgaga atagtgtatg cggcgaccga
     9841 gttgctcttg cccggcgtca atacgggata ataccgcgcc acatagcaga actttaaaag
     9901 tgctcatcat tggaaaacgt tcttcggggc gaaaactctc aaggatctta ccgctgttga
     9961 gatccagttc gatgtaaccc actcgtgcac ccaactgatc ttcagcatct tttactttca
    10021 ccagcgtttc tgggtgagca aaaacaggaa ggcaaaatgc cgcaaaaaag ggaataaggg
    10081 cgacacggaa atgttgaata ctcatactct tcctttttca atattattga agcatttatc
    10141 agggttattg tctcatgagc ggatacatat ttgaatgtat ttagaaaaat aaacaaatag
    10201 gggttccgcg cacatttccc cgaaaagtgc cacctgacgt cgacggatcg ggagatctcc
    10261 cgatccccta tggtgcactc tcagtacaat ctgctctgat gccgcatagt taagccagta
    10321 tctgctccct gcttgtgtgt tggaggtcgc tgagtagtgc gcgagcaaaa tttaagctac
    10381 aacaaggcaa ggcttgaccg acaattgcat gaagaatctg cttagggtta ggcgttttgc
    10441 gctgcttcgc gatgtacggg ccagatatac gcgttgacat tgattattga ctagttatta
    10501 atagtaatca attacggggt cattagttca tagcccatat atggagttcc gcgttacata
    10561 acttacggta aatggcccgc ctggctgacc gcccaacgac ccccgcccat tgacgtcaat
    10621 aatgacgtat gttcccatag taacgccaat agggactttc cattgacgtc aatgggtgga
    10681 gtatttacgg taaactgccc acttggcagt acatcaagtg tatcatatgc caagtacgcc
    10741 ccctattgac gtcaatgacg gtaaatggcc cgcctggcat tatgcccagt acatgacctt
    10801 atgggacttt cctacttggc agtacatcta cgtattagtc atcgctatta ccatggtgat
    10861 gcggttttgg cagtacatca atgggcgtgg atagcggttt gactcacggg gatttccaag
    10921 tctccacccc attgacgtca atgggagttt gttttggcac caaaatcaac gggactttcc
    10981 aaaatgtcgt aacaactccg ccccattgac gcaaatgggc ggtaggcgtg tacggtggga
    11041 ggtctatata agcagcgcgt tttgcctgta ctgggtctct ctggttagac cagatctgag
    11101 cctgggagct ctctggctaa ctagggaacc cactgcttaa gcctcaataa agcttgcctt
    11161 gagtgcttca agtagtgtgt gcccgtctgt tgtgtgactc tggtaactag agatccctca
    11221 gaccctttta gtcagtgtgg aaaatctcta gcagtggcgc ccgaacaggg acttgaaagc
    11281 gaaagggaaa ccagaggagc tctctcgacg caggactcgg cttgctgaag cgcgcacggc
    11341 aagaggcgag gggcggcgac tggtgagtac gccaaaaatt ttgactagcg gaggctagaa
    11401 ggagagagat gggtgcgaga gcgtcagtat taagcggggg agaattagat cgcgatggga
    11461 aaaaattcgg ttaaggccag ggggaaagaa aaaatataaa ttaaaacata tagtatgggc
    11521 aagcagggag ctagaacgat tcgcagttaa tcctggcctg ttagaaacat cagaaggctg
    11581 tagacaaata ctgggacagc tacaaccatc ccttcagaca ggatcagaag aacttagatc
    11641 attatataat acagtagcaa ccctctattg tgtgcatcaa aggatagaga taaaagacac
    11701 caaggaagct ttagacaaga tagaggaaga gcaaaacaaa agtaagacca ccgcacagca
    11761 agcggccgct gatcttcaga cctggaggag gagatatgag ggacaattgg agaagtgaat
    11821 tatataaata taaagtagta aaaattgaac cattaggagt agcacccacc aaggcaaaga
    11881 gaagagtggt gcagagagaa aaaagagcag tgggaatagg agctttgttc cttgggttct
    11941 tgggagcagc aggaagcact atgggcgcag cgtcaatgac gctgacggta caggccagac
    12001 aattattgtc tggtatagtg cagcagcaga acaatttgct gagggctatt gaggcgcaac
    12061 agcatctgtt gcaactcaca gtctggggca tcaagcagct ccaggcaaga atcctggctg
    12121 tggaaagata cctaaaggat caacagctcc tggggatttg gggttgctct ggaaaactca
    12181 tttgcaccac tgctgtgcct tggaatgcta gttggagtaa taaatctctg gaacagattt
    12241 ggaatcacac gacctggatg gagtgggaca gagaaattaa caattacaca agcttaatac
    12301 actccttaat tgaagaatcg caaaaccagc aagaaaagaa tgaacaagaa ttattggaat
    12361 tagataaatg ggcaagtttg tggaattggt ttaacataac aaattggctg tggtatataa
    12421 aattattcat aatgatagta ggaggcttgg taggtttaag aatagttttt gctgtacttt
    12481 ctatagtgaa tagagttagg cagggatatt caccattatc gtttcagacc cacctcccaa
    12541 ccccgagggg acccgacagg cccgaaggaa tagaagaaga aggtggagag agagacagag
    12601 acagatccat tcgattagtg aacggatcgg cactgcgtgc gccaattctg cagacaaatg
    12661 gcagtattca tccacaattt taaaagaaaa ggggggattg gggggtacag tgcaggggaa
    12721 agaatagtag acataatagc aacagacata caaactaaag aattacaaaa acaaattaca
    12781 aaaattcaaa attttcgggt ttattacagg gacagcagag atccagtttg gttaattaag
    12841 gtaccgaggg cctatttccc atgattcctt catatttgca tatacgatac aaggctgtta
    12901 gagagataat tagaattaat ttgactgtaa acacaaagat attagtacaa aatacgtgac
    12961 gtagaaagta ataatttctt gggtagtttg cagttttaaa attatgtttt aaaatggact
    13021 atcatatgct taccgtaact tgaaagtatt tcgatttctt ggctttatat atcttgtgga
    13081 aaggacgaaa caccgtacgt tctctatcac tgat
//
