LOCUS       sequence-389756-       12437 bp    DNA     circular SYN 15-DEC-2025
DEFINITION  Tet-inducible expression of Cas9-T2A-TagBFP.
ACCESSION   .
VERSION     .
KEYWORDS    .
SOURCE      synthetic DNA construct
  ORGANISM  synthetic DNA construct
REFERENCE   1  (bases 1 to 12437)
  TITLE     Schmierer unpublished plasmids
REFERENCE   2  (bases 1 to 12437)
  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: pCW-Tet-Cas9-BFP-PGK-Blast
FEATURES             Location/Qualifiers
     source          1..12437
                     /mol_type="other DNA"
                     /organism="synthetic DNA construct"
     LTR             1..181
                     /label=5' LTR (truncated)
                     /note="truncated 5' long terminal repeat (LTR) from HIV-1"
     misc_feature    228..353
                     /label=HIV-1 Psi
                     /note="packaging signal of human immunodeficiency virus 
                     type 1"
     misc_feature    846..1079
                     /label=RRE
                     /note="The Rev response element (RRE) of HIV-1 allows for 
                     Rev-dependent mRNA export from the nucleus to the 
                     cytoplasm."
     CDS             1264..1308
                     /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    1521..1638
                     /label=cPPT/CTS
                     /note="central polypurine tract and central termination 
                     sequence of HIV-1"
     promoter        1692..2006
                     /label=tight TRE promoter
                     /note="Tet-responsive promoter PTight, consisting of seven 
                     tet operator sequences followed by the minimal CMV 
                     promoter"
     protein_bind    1700..1718
                     /gene="tetO"
                     /label=tet operator
                     /bound_moiety="tetracycline repressor TetR"
                     /note="bacterial operator O2 for the tetR and tetA genes"
     protein_bind    1736..1754
                     /gene="tetO"
                     /label=tet operator
                     /bound_moiety="tetracycline repressor TetR"
                     /note="bacterial operator O2 for the tetR and tetA genes"
     protein_bind    1771..1789
                     /gene="tetO"
                     /label=tet operator
                     /bound_moiety="tetracycline repressor TetR"
                     /note="bacterial operator O2 for the tetR and tetA genes"
     protein_bind    1807..1825
                     /gene="tetO"
                     /label=tet operator
                     /bound_moiety="tetracycline repressor TetR"
                     /note="bacterial operator O2 for the tetR and tetA genes"
     protein_bind    1843..1861
                     /gene="tetO"
                     /label=tet operator
                     /bound_moiety="tetracycline repressor TetR"
                     /note="bacterial operator O2 for the tetR and tetA genes"
     protein_bind    1878..1896
                     /gene="tetO"
                     /label=tet operator
                     /bound_moiety="tetracycline repressor TetR"
                     /note="bacterial operator O2 for the tetR and tetA genes"
     protein_bind    1914..1932
                     /gene="tetO"
                     /label=tet operator
                     /bound_moiety="tetracycline repressor TetR"
                     /note="bacterial operator O2 for the tetR and tetA genes"
     primer_bind     1979..2003
                     /label=LNCX
                     /note="Human CMV promoter, forward primer"
     regulatory      2023..2032
                     /label=Kozak sequence
                     /note="vertebrate consensus sequence for strong initiation 
                     of translation (Kozak, 1987)"
                     /regulatory_class="other"
     CDS             2032..2097
                     /codon_start=1
                     /product="three tandem FLAG(R) epitope tags, followed by an
                     enterokinase cleavage site"
                     /label=3xFLAG
                     /translation="DYKDHDGDYKDHDIDYKDDDDK"
     CDS             2104..2124
                     /codon_start=1
                     /product="nuclear localization signal of SV40 (simian virus
                     40) large T antigen"
                     /label=SV40 NLS
                     /translation="PKKKRKV"
     CDS             2149..6249
                     /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="DKKYSIGLDIGTNSVGWAVITDEYKVPSKKFKVLGNTDRHSIKKN
                     LIGALLFDSGETAEATRLKRTARRRYTRRKNRICYLQEIFSNEMAKVDDSFFHRLEESF
                     LVEEDKKHERHPIFGNIVDEVAYHEKYPTIYHLRKKLVDSTDKADLRLIYLALAHMIKF
                     RGHFLIEGDLNPDNSDVDKLFIQLVQTYNQLFEENPINASGVDAKAILSARLSKSRRLE
                     NLIAQLPGEKKNGLFGNLIALSLGLTPNFKSNFDLAEDAKLQLSKDTYDDDLDNLLAQI
                     GDQYADLFLAAKNLSDAILLSDILRVNTEITKAPLSASMIKRYDEHHQDLTLLKALVRQ
                     QLPEKYKEIFFDQSKNGYAGYIDGGASQEEFYKFIKPILEKMDGTEELLVKLNREDLLR
                     KQRTFDNGSIPHQIHLGELHAILRRQEDFYPFLKDNREKIEKILTFRIPYYVGPLARGN
                     SRFAWMTRKSEETITPWNFEEVVDKGASAQSFIERMTNFDKNLPNEKVLPKHSLLYEYF
                     TVYNELTKVKYVTEGMRKPAFLSGEQKKAIVDLLFKTNRKVTVKQLKEDYFKKIECFDS
                     VEISGVEDRFNASLGTYHDLLKIIKDKDFLDNEENEDILEDIVLTLTLFEDREMIEERL
                     KTYAHLFDDKVMKQLKRRRYTGWGRLSRKLINGIRDKQSGKTILDFLKSDGFANRNFMQ
                     LIHDDSLTFKEDIQKAQVSGQGDSLHEHIANLAGSPAIKKGILQTVKVVDELVKVMGRH
                     KPENIVIEMARENQTTQKGQKNSRERMKRIEEGIKELGSQILKEHPVENTQLQNEKLYL
                     YYLQNGRDMYVDQELDINRLSDYDVDHIVPQSFLKDDSIDNKVLTRSDKNRGKSDNVPS
                     EEVVKKMKNYWRQLLNAKLITQRKFDNLTKAERGGLSELDKAGFIKRQLVETRQITKHV
                     AQILDSRMNTKYDENDKLIREVKVITLKSKLVSDFRKDFQFYKVREINNYHHAHDAYLN
                     AVVGTALIKKYPKLESEFVYGDYKVYDVRKMIAKSEQEIGKATAKYFFYSNIMNFFKTE
                     ITLANGEIRKRPLIETNGETGEIVWDKGRDFATVRKVLSMPQVNIVKKTEVQTGGFSKE
                     SILPKRNSDKLIARKKDWDPKKYGGFDSPTVAYSVLVVAKVEKGKSKKLKSVKELLGIT
                     IMERSSFEKNPIDFLEAKGYKEVKKDLIIKLPKYSLFELENGRKRMLASAGELQKGNEL
                     ALPSKYVNFLYLASHYEKLKGSPEDNEQKQLFVEQHKHYLDEIIEQISEFSKRVILADA
                     NLDKVLSAYNKHRDKPIREQAENIIHLFTLTNLGAPAAFKYFDTTIDRKRYTSTKEVLD
                     ATLIHQSITGLYETRIDLSQLGGD"
     CDS             2149..6249
                     /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="DKKYSIGLDIGTNSVGWAVITDEYKVPSKKFKVLGNTDRHSIKKN
                     LIGALLFDSGETAEATRLKRTARRRYTRRKNRICYLQEIFSNEMAKVDDSFFHRLEESF
                     LVEEDKKHERHPIFGNIVDEVAYHEKYPTIYHLRKKLVDSTDKADLRLIYLALAHMIKF
                     RGHFLIEGDLNPDNSDVDKLFIQLVQTYNQLFEENPINASGVDAKAILSARLSKSRRLE
                     NLIAQLPGEKKNGLFGNLIALSLGLTPNFKSNFDLAEDAKLQLSKDTYDDDLDNLLAQI
                     GDQYADLFLAAKNLSDAILLSDILRVNTEITKAPLSASMIKRYDEHHQDLTLLKALVRQ
                     QLPEKYKEIFFDQSKNGYAGYIDGGASQEEFYKFIKPILEKMDGTEELLVKLNREDLLR
                     KQRTFDNGSIPHQIHLGELHAILRRQEDFYPFLKDNREKIEKILTFRIPYYVGPLARGN
                     SRFAWMTRKSEETITPWNFEEVVDKGASAQSFIERMTNFDKNLPNEKVLPKHSLLYEYF
                     TVYNELTKVKYVTEGMRKPAFLSGEQKKAIVDLLFKTNRKVTVKQLKEDYFKKIECFDS
                     VEISGVEDRFNASLGTYHDLLKIIKDKDFLDNEENEDILEDIVLTLTLFEDREMIEERL
                     KTYAHLFDDKVMKQLKRRRYTGWGRLSRKLINGIRDKQSGKTILDFLKSDGFANRNFMQ
                     LIHDDSLTFKEDIQKAQVSGQGDSLHEHIANLAGSPAIKKGILQTVKVVDELVKVMGRH
                     KPENIVIEMARENQTTQKGQKNSRERMKRIEEGIKELGSQILKEHPVENTQLQNEKLYL
                     YYLQNGRDMYVDQELDINRLSDYDVDHIVPQSFLKDDSIDNKVLTRSDKNRGKSDNVPS
                     EEVVKKMKNYWRQLLNAKLITQRKFDNLTKAERGGLSELDKAGFIKRQLVETRQITKHV
                     AQILDSRMNTKYDENDKLIREVKVITLKSKLVSDFRKDFQFYKVREINNYHHAHDAYLN
                     AVVGTALIKKYPKLESEFVYGDYKVYDVRKMIAKSEQEIGKATAKYFFYSNIMNFFKTE
                     ITLANGEIRKRPLIETNGETGEIVWDKGRDFATVRKVLSMPQVNIVKKTEVQTGGFSKE
                     SILPKRNSDKLIARKKDWDPKKYGGFDSPTVAYSVLVVAKVEKGKSKKLKSVKELLGIT
                     IMERSSFEKNPIDFLEAKGYKEVKKDLIIKLPKYSLFELENGRKRMLASAGELQKGNEL
                     ALPSKYVNFLYLASHYEKLKGSPEDNEQKQLFVEQHKHYLDEIIEQISEFSKRVILADA
                     NLDKVLSAYNKHRDKPIREQAENIIHLFTLTNLGAPAAFKYFDTTIDRKRYTSTKEVLD
                     ATLIHQSITGLYETRIDLSQLGGD"
     CDS             6250..6297
                     /codon_start=1
                     /product="bipartite nuclear localization signal from 
                     nucleoplasmin"
                     /label=nucleoplasmin NLS
                     /translation="KRPAATKKAGQAKKKK"
     CDS             6307..6360
                     /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             6361..7059
                     /codon_start=1
                     /product="monomeric blue fluorescent protein"
                     /label=TagBFP
                     /note="mammalian codon-optimized"
                     /translation="SELIKENMHMKLYMEGTVDNHHFKCTSEGEGKPYEGTQTMRIKVV
                     EGGPLPFAFDILATSFLYGSKTFINHTQGIPDFFKQSFPEGFTWERVTTYEDGGVLTAT
                     QDTSLQDGCLIYNVKIRGVNFTSNGPVMQKKTLGWEAFTETLYPADGGLEGRNDMALKL
                     VGGSHLIANIKTTYRSKKPAKNLKMPGVYYVDYRLERIKEANNETYVEQHEVAVARYCD
                     LPSKLGHKLN"
     promoter        7060..7570
                     /label=hPGK promoter
                     /note="human phosphoglycerate kinase 1 promoter"
     primer_bind     7489..7507
                     /label=hPGK-F
                     /note="Human PGK promoter, forward primer"
     CDS             7580..7975
                     /codon_start=1
                     /gene="Aspergillus terreus BSD"
                     /product="blasticidin S deaminase"
                     /label=BSD
                     /note="confers resistance to blasticidin"
                     /translation="MAKPLSQEESTLIERATATINSIPISEDYSVASAALSSDGRIFTG
                     VNVYHFTGGPCAELVVLGTAAAAAAGNLTCIVAIGNENRGILSPCGRCRQVLLDLHPGI
                     KAIVKDSDGQPTAVGIRELLPSGYVWEG"
     CDS             7976..8029
                     /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             join(8030..8647,8648..8653,8654..8689,8690..8692,8693..8728,
                     8729..8731,8732..8767,8768..8776)
                     /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
                     KNKRALLDALPIEMLDRHHTHFCPLEGESWQDFLRNNAKSYRCALLSHRDGAKVHLGTR
                     PTEKQYETLENQLAFLCQQGFSLENALYALSAVGHFTLGCVLEEQEHQVAKEERETPTT
                     DSMPPLLRQAIELFDRQGAEPAFLFGLELIICGLEKQLKCESGGPTDALDDFDLDMLPA
                     DALDDFDLDMLPADALDDFDLDMLPG"
     primer_bind     complement(8816..8836)
                     /label=WPRE-R
                     /note="WPRE, reverse primer"
     CDS             complement(9234..9245)
                     /codon_start=1
                     /product="Factor Xa recognition and cleavage site"
                     /label=Factor Xa site
                     /translation="IEGR"
     LTR             9450..9683
                     /label=3' LTR (Delta-U3)
                     /note="self-inactivating 3' long terminal repeat (LTR) from
                     HIV-1"
     polyA_signal    9755..9876
                     /label=SV40 poly(A) signal
                     /note="SV40 polyadenylation signal"
     primer_bind     complement(9792..9811)
                     /label=SV40pA-R
                     /note="SV40 polyA, reverse primer"
     primer_bind     9846..9865
                     /label=EBV-rev
                     /note="SV40 polyA terminator, reverse primer"
     rep_origin      9916..10051
                     /label=SV40 ori
                     /note="SV40 origin of replication"
     promoter        10051..10122
                     /gene="bla"
                     /label=AmpR promoter
     CDS             join(10123..10191,10192..10983)
                     /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(10341..10360)
                     /label=Amp-R
                     /note="Ampicillin resistance gene, reverse primer"
     rep_origin      11154..11742
                     /direction=RIGHT
                     /label=ori
                     /note="high-copy-number ColE1/pMB1/pBR322/pUC origin of 
                     replication"
     primer_bind     11643..11662
                     /label=pBR322ori-F
                     /note="pBR322 origin, forward primer"
     primer_bind     11896..11913
                     /label=L4440
                     /note="L4440 vector, forward primer"
     protein_bind    12030..12051
                     /label=CAP binding site
                     /bound_moiety="E. coli catabolite activator protein"
                     /note="CAP binding activates transcription in the presence 
                     of cAMP."
     promoter        join(12066..12071,12072..12089,12090..12096)
                     /label=lac promoter
                     /note="promoter for the E. coli lac operon"
     protein_bind    12104..12120
                     /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     12109..12131
                     /label=M13/pUC Reverse
                     /note="In lacZ gene"
     primer_bind     12128..12144
                     /label=M13 rev
                     /note="common sequencing primer, one of multiple similar 
                     variants"
     primer_bind     12128..12144
                     /label=M13 Reverse
                     /note="In lacZ gene. Also called M13-rev"
     primer_bind     12163..12183
                     /label=T3
                     /note="T3 promoter, forward primer"
     promoter        12165..12183
                     /label=T3 promoter
                     /note="promoter for bacteriophage T3 RNA polymerase"
     promoter        12211..12437
                     /label=RSV promoter
                     /note="Rous sarcoma virus enhancer/promoter"
ORIGIN
        1 gggtctctct ggttagacca gatctgagcc tgggagctct ctggctaact agggaaccca
       61 ctgcttaagc ctcaataaag cttgccttga gtgcttcaag tagtgtgtgc ccgtctgttg
      121 tgtgactctg gtaactagag atccctcaga cccttttagt cagtgtggaa aatctctagc
      181 agtggcgccc gaacagggac ttgaaagcga aagggaaacc agaggagctc tctcgacgca
      241 ggactcggct tgctgaagcg cgcacggcaa gaggcgaggg gcggcgactg gtgagtacgc
      301 caaaaatttt gactagcgga ggctagaagg agagagatgg gtgcgagagc gtcagtatta
      361 agcgggggag aattagatcg cgatgggaaa aaattcggtt aaggccaggg ggaaagaaaa
      421 aatataaatt aaaacatata gtatgggcaa gcagggagct agaacgattc gcagttaatc
      481 ctggcctgtt agaaacatca gaaggctgta gacaaatact gggacagcta caaccatccc
      541 ttcagacagg atcagaagaa cttagatcat tatataatac agtagcaacc ctctattgtg
      601 tgcatcaaag gatagagata aaagacacca aggaagcttt agacaagata gaggaagagc
      661 aaaacaaaag taagaccacc gcacagcaag cggccgctga tcttcagacc tggaggagga
      721 gatatgaggg acaattggag aagtgaatta tataaatata aagtagtaaa aattgaacca
      781 ttaggagtag cacccaccaa ggcaaagaga agagtggtgc agagagaaaa aagagcagtg
      841 ggaataggag ctttgttcct tgggttcttg ggagcagcag gaagcactat gggcgcagcg
      901 tcaatgacgc tgacggtaca ggccagacaa ttattgtctg gtatagtgca gcagcagaac
      961 aatttgctga gggctattga ggcgcaacag catctgttgc aactcacagt ctggggcatc
     1021 aagcagctcc aggcaagaat cctggctgtg gaaagatacc taaaggatca acagctcctg
     1081 gggatttggg gttgctctgg aaaactcatt tgcaccactg ctgtgccttg gaatgctagt
     1141 tggagtaata aatctctgga acagatttgg aatcacacga cctggatgga gtgggacaga
     1201 gaaattaaca attacacaag cttaatacac tccttaattg aagaatcgca aaaccagcaa
     1261 gaaaagaatg aacaagaatt attggaatta gataaatggg caagtttgtg gaattggttt
     1321 aacataacaa attggctgtg gtatataaaa ttattcataa tgatagtagg aggcttggta
     1381 ggtttaagaa tagtttttgc tgtactttct atagtgaata gagttaggca gggatattca
     1441 ccattatcgt ttcagaccca cctcccaacc ccgaggggac aattctcgac ctcgagacaa
     1501 atggcagtat tcatccacaa ttttaaaaga aaagggggga ttggggggta cagtgcaggg
     1561 gaaagaatag tagacataat agcaacagac atacaaacta aagaattaca aaaacaaatt
     1621 acaaaaattc aaaattttcg ggtttattac agggacagca gagatccact ttggccgcga
     1681 atcgatatgt cgagtttact ccctatcagt gatagagaac gtatgtcgag tttactccct
     1741 atcagtgata gagaacgatg tcgagtttac tccctatcag tgatagagaa cgtatgtcga
     1801 gtttactccc tatcagtgat agagaacgta tgtcgagttt actccctatc agtgatagag
     1861 aacgtatgtc gagtttatcc ctatcagtga tagagaacgt atgtcgagtt tactccctat
     1921 cagtgataga gaacgtatgt cgaggtaggc gtgtacggtg ggaggcctat ataagcagag
     1981 ctcgtttagt gaaccgtcag atcgcctgga gaattggcta gcgccaccat ggactataag
     2041 gaccacgacg gagactacaa ggatcatgat attgattaca aagacgatga cgataagatg
     2101 gccccaaaga agaagcggaa ggtcggtatc cacggagtcc cagcagccga caagaagtac
     2161 agcatcggcc tggacatcgg caccaactct gtgggctggg ccgtgatcac cgacgagtac
     2221 aaggtgccca gcaagaaatt caaggtgctg ggcaacaccg accggcacag catcaagaag
     2281 aacctgatcg gagccctgct gttcgacagc ggcgaaacag ccgaggccac ccggctgaag
     2341 agaaccgcca gaagaagata caccagacgg aagaaccgga tctgctatct gcaagagatc
     2401 ttcagcaacg agatggccaa ggtggacgac agcttcttcc acagactgga agagtccttc
     2461 ctggtggaag aggataagaa gcacgagcgg caccccatct tcggcaacat cgtggacgag
     2521 gtggcctacc acgagaagta ccccaccatc taccacctga gaaagaaact ggtggacagc
     2581 accgacaagg ccgacctgcg gctgatctat ctggccctgg cccacatgat caagttccgg
     2641 ggccacttcc tgatcgaggg cgacctgaac cccgacaaca gcgacgtgga caagctgttc
     2701 atccagctgg tgcagaccta caaccagctg ttcgaggaaa accccatcaa cgccagcggc
     2761 gtggacgcca aggccatcct gtctgccaga ctgagcaaga gcagacggct ggaaaatctg
     2821 atcgcccagc tgcccggcga gaagaagaat ggcctgttcg gaaacctgat tgccctgagc
     2881 ctgggcctga cccccaactt caagagcaac ttcgacctgg ccgaggatgc caaactgcag
     2941 ctgagcaagg acacctacga cgacgacctg gacaacctgc tggcccagat cggcgaccag
     3001 tacgccgacc tgtttctggc cgccaagaac ctgtccgacg ccatcctgct gagcgacatc
     3061 ctgagagtga acaccgagat caccaaggcc cccctgagcg cctctatgat caagagatac
     3121 gacgagcacc accaggacct gaccctgctg aaagctctcg tgcggcagca gctgcctgag
     3181 aagtacaaag agattttctt cgaccagagc aagaacggct acgccggcta cattgacggc
     3241 ggagccagcc aggaagagtt ctacaagttc atcaagccca tcctggaaaa gatggacggc
     3301 accgaggaac tgctcgtgaa gctgaacaga gaggacctgc tgcggaagca gcggaccttc
     3361 gacaacggca gcatccccca ccagatccac ctgggagagc tgcacgccat tctgcggcgg
     3421 caggaagatt tttacccatt cctgaaggac aaccgggaaa agatcgagaa gatcctgacc
     3481 ttccgcatcc cctactacgt gggccctctg gccaggggaa acagcagatt cgcctggatg
     3541 accagaaaga gcgaggaaac catcaccccc tggaacttcg aggaagtggt ggacaagggc
     3601 gcttccgccc agagcttcat cgagcggatg accaacttcg ataagaacct gcccaacgag
     3661 aaggtgctgc ccaagcacag cctgctgtac gagtacttca ccgtgtataa cgagctgacc
     3721 aaagtgaaat acgtgaccga gggaatgaga aagcccgcct tcctgagcgg cgagcagaaa
     3781 aaggccatcg tggacctgct gttcaagacc aaccggaaag tgaccgtgaa gcagctgaaa
     3841 gaggactact tcaagaaaat cgagtgcttc gactccgtgg aaatctccgg cgtggaagat
     3901 cggttcaacg cctccctggg cacataccac gatctgctga aaattatcaa ggacaaggac
     3961 ttcctggaca atgaggaaaa cgaggacatt ctggaagata tcgtgctgac cctgacactg
     4021 tttgaggaca gagagatgat cgaggaacgg ctgaaaacct atgcccacct gttcgacgac
     4081 aaagtgatga agcagctgaa gcggcggaga tacaccggct ggggcaggct gagccggaag
     4141 ctgatcaacg gcatccggga caagcagtcc ggcaagacaa tcctggattt cctgaagtcc
     4201 gacggcttcg ccaacagaaa cttcatgcag ctgatccacg acgacagcct gacctttaaa
     4261 gaggacatcc agaaagccca ggtgtccggc cagggcgata gcctgcacga gcacattgcc
     4321 aatctggccg gcagccccgc cattaagaag ggcatcctgc agacagtgaa ggtggtggac
     4381 gagctcgtga aagtgatggg ccggcacaag cccgagaaca tcgtgatcga aatggccaga
     4441 gagaaccaga ccacccagaa gggacagaag aacagccgcg agagaatgaa gcggatcgaa
     4501 gagggcatca aagagctggg cagccagatc ctgaaagaac accccgtgga aaacacccag
     4561 ctgcagaacg agaagctgta cctgtactac ctgcagaatg ggcgggatat gtacgtggac
     4621 caggaactgg acatcaaccg gctgtccgac tacgatgtgg accatatcgt gcctcagagc
     4681 tttctgaagg acgactccat cgacaacaag gtgctgacca gaagcgacaa gaaccggggc
     4741 aagagcgaca acgtgccctc cgaagaggtc gtgaagaaga tgaagaacta ctggcggcag
     4801 ctgctgaacg ccaagctgat tacccagaga aagttcgaca atctgaccaa ggccgagaga
     4861 ggcggcctga gcgaactgga taaggccggc ttcatcaaga gacagctggt ggaaacccgg
     4921 cagatcacaa agcacgtggc acagatcctg gactcccgga tgaacactaa gtacgacgag
     4981 aatgacaagc tgatccggga agtgaaagtg atcaccctga agtccaagct ggtgtccgat
     5041 ttccggaagg atttccagtt ttacaaagtg cgcgagatca acaactacca ccacgcccac
     5101 gacgcctacc tgaacgccgt cgtgggaacc gccctgatca aaaagtaccc taagctggaa
     5161 agcgagttcg tgtacggcga ctacaaggtg tacgacgtgc ggaagatgat cgccaagagc
     5221 gagcaggaaa tcggcaaggc taccgccaag tacttcttct acagcaacat catgaacttt
     5281 ttcaagaccg agattaccct ggccaacggc gagatccgga agcggcctct gatcgagaca
     5341 aacggcgaaa ccggggagat cgtgtgggat aagggccggg attttgccac cgtgcggaaa
     5401 gtgctgagca tgccccaagt gaatatcgtg aaaaagaccg aggtgcagac aggcggcttc
     5461 agcaaagagt ctatcctgcc caagaggaac agcgataagc tgatcgccag aaagaaggac
     5521 tgggacccta agaagtacgg cggcttcgac agccccaccg tggcctattc tgtgctggtg
     5581 gtggccaaag tggaaaaggg caagtccaag aaactgaaga gtgtgaaaga gctgctgggg
     5641 atcaccatca tggaaagaag cagcttcgag aagaatccca tcgactttct ggaagccaag
     5701 ggctacaaag aagtgaaaaa ggacctgatc atcaagctgc ctaagtactc cctgttcgag
     5761 ctggaaaacg gccggaagag aatgctggcc tctgccggcg aactgcagaa gggaaacgaa
     5821 ctggccctgc cctccaaata tgtgaacttc ctgtacctgg ccagccacta tgagaagctg
     5881 aagggctccc ccgaggataa tgagcagaaa cagctgtttg tggaacagca caagcactac
     5941 ctggacgaga tcatcgagca gatcagcgag ttctccaaga gagtgatcct ggccgacgct
     6001 aatctggaca aagtgctgtc cgcctacaac aagcaccggg ataagcccat cagagagcag
     6061 gccgagaata tcatccacct gtttaccctg accaatctgg gagcccctgc cgccttcaag
     6121 tactttgaca ccaccatcga ccggaagagg tacaccagca ccaaagaggt gctggacgcc
     6181 accctgatcc accagagcat caccggcctg tacgagacac ggatcgacct gtctcagctg
     6241 ggaggcgaca aaaggccggc ggccacgaaa aaggccggcc aggcaaaaaa gaaaaagggc
     6301 agtggagagg gcagaggaag tctgctaaca tgcggtgacg tcgaggagaa tcctggccca
     6361 agcgagctga ttaaggagaa catgcacatg aagctctaca tggagggcac cgtggacaac
     6421 catcacttca agtgcacatc cgagggcgaa ggcaagccct acgagggcac ccagaccatg
     6481 agaatcaagg tggtcgaggg cggccctctc cccttcgcct tcgacatcct ggctactagc
     6541 ttcctctacg gcagcaagac cttcatcaac cacacccagg gcatccccga cttcttcaag
     6601 cagtccttcc ctgagggctt cacatgggag agagtcacca catacgaaga cgggggcgtg
     6661 ctgaccgcta cccaggacac cagcctccag gacggctgcc tcatctacaa cgtcaagatc
     6721 agaggggtga acttcacatc caacggccct gtgatgcaga agaaaacact cggctgggag
     6781 gccttcaccg aaacgctgta ccccgctgac ggcggcctgg aaggcagaaa cgacatggcc
     6841 ctgaagctcg tgggcgggag ccatctgatc gcaaacatca agaccacata tagatccaag
     6901 aaacccgcta agaacctcaa gatgcctggc gtctactatg tggactacag actggaaaga
     6961 atcaaggagg ccaacaacga gacctacgtc gagcagcacg aggtggcagt ggccagatac
     7021 tgcgacctcc ctagcaaact ggggcacaag cttaattaag gggttggggt tgcgcctttt
     7081 ccaaggcagc cctgggtttg cgcagggacg cggctgctct gggcgtggtt ccgggaaacg
     7141 cagcggcgcc gaccctgggt ctcgcacatt cttcacgtcc gttcgcagcg tcacccggat
     7201 cttcgccgct acccttgtgg gccccccggc gacgcttcct gctccgcccc taagtcggga
     7261 aggttccttg cggttcgcgg cgtgccggac gtgacaaacg gaagccgcac gtctcactag
     7321 taccctcgca gacggacagc gccagggagc aatggcagcg cgccgaccgc gatgggctgt
     7381 ggccaatagc ggctgctcag cagggcgcgc cgagagcagc ggccgggaag gggcggtgcg
     7441 ggaggcgggg tgtggggcgg tagtgtgggc cctgttcctg cccgcgcggt gttccgcatt
     7501 ctgcaagcct ccggagcgca cgtcggcagt cggctccctc gttgaccgaa tcaccgacct
     7561 ctctccccag caattcacca tggccaagcc tttgtctcaa gaagaatcca ccctcattga
     7621 aagagcaacg gctacaatca acagcatccc catctctgaa gactacagcg tcgccagcgc
     7681 agctctctct agcgacggcc gcatcttcac tggtgtcaat gtatatcatt ttactggggg
     7741 accttgtgca gaactcgtgg tgctgggcac tgctgctgct gcggcagctg gcaacctgac
     7801 ttgtatcgtc gcgatcggaa atgagaacag gggcatcttg agcccctgcg gacggtgccg
     7861 acaggtgctt ctcgatctgc atcctgggat caaagccata gtgaaggaca gtgatggaca
     7921 gccgacggca gttgggattc gtgaattgct gccctctggt tatgtgtggg agggcgaagg
     7981 tagaggttct ctcctcactt gtggtgatgt tgaagaaaac cctggtccaa tgtctagact
     8041 ggacaagagc aaagtcataa acggagctct ggaattactc aatggtgtcg gtatcgaagg
     8101 cctgacgaca aggaaactcg ctcaaaagct gggagttgag cagcctaccc tgtactggca
     8161 cgtgaagaac aagcgggccc tgctcgatgc cctgccaatc gagatgctgg acaggcatca
     8221 tacccacttc tgccccctgg aaggcgagtc atggcaagac tttctgcgga acaacgccaa
     8281 gtcataccgc tgtgctctcc tctcacatcg cgacggggct aaagtgcatc tcggcacccg
     8341 cccaacagag aaacagtacg aaaccctgga aaatcagctc gcgttcctgt gtcagcaagg
     8401 cttctccctg gagaacgcac tgtacgctct gtccgccgtg ggccacttta cactgggctg
     8461 cgtattggag gaacaggagc atcaagtagc aaaagaggaa agagagacac ctaccaccga
     8521 ttctatgccc ccacttctga gacaagcaat tgagctgttc gaccggcagg gagccgaacc
     8581 tgccttcctt ttcggcctgg aactaatcat atgtggcctg gagaaacagc taaagtgcga
     8641 aagcggcggg ccgaccgacg cccttgacga ttttgactta gacatgctcc cagccgatgc
     8701 ccttgacgac tttgaccttg atatgctgcc tgctgacgct cttgacgatt ttgaccttga
     8761 catgctcccc gggtaactaa gtaaggatcg atccaagata tcgtattctt aactatgttg
     8821 ctccttttac gctatgtgga tacgctgctt taatgccttt gtatcatgct attgcttccc
     8881 gtatggcttt cattttctcc tccttgtata aatcctggtt gctgtctctt tatgaggagt
     8941 tgtggcccgt tgtcaggcaa cgtggcgtgg tgtgcactgt gtttgctgac gcaaccccca
     9001 ctggttgggg cattgccacc acctgtcagc tcctttccgg gactttcgct ttccccctcc
     9061 ctattgccac ggcggaactc atcgccgcct gccttgcccg ctgctggaca ggggctcggc
     9121 tgttgggcac tgacaattcc gtggtgttgt cggggaagct gacgtccttt ccatggctgc
     9181 tcgcctgtgt tgccacctgg attctgcgcg ggacgtcctt ctgctacgtc ccttcggccc
     9241 tcaatccagc ggaccttcct tcccgcggcc tgctgccggc tctgcggcct cttccgcgtc
     9301 ttcgccttcg ccctcagacg agtcggatct ccctttgggc cgcctccccg cctgtttcgc
     9361 ctcggcgtcc ggactagagg tacctttaag accaatgact tacaaggcag ctgtagatct
     9421 tagccacttt ttaaaagaaa aggggggact ggaagggcta attcactccc aacgaagaca
     9481 agatctgctt tttgcttgta ctgggtctct ctggttagac cagatctgag cctgggagct
     9541 ctctggctaa ctagggaacc cactgcttaa gcctcaataa agcttgcctt gagtgcttca
     9601 agtagtgtgt gcccgtctgt tgtgtgactc tggtaactag agatccctca gaccctttta
     9661 gtcagtgtgg aaaatctcta gcagtagtag ttcatgtcat cttattattc agtatttata
     9721 acttgcaaag aaatgaatat cagagagtga gaggaacttg tttattgcag cttataatgg
     9781 ttacaaataa agcaatagca tcacaaattt cacaaataaa gcattttttt cactgcattc
     9841 tagttgtggt ttgtccaaac tcatcaatgt atcttatcat gtctggctct agctatcccg
     9901 cccctaactc cgcccatccc gcccctaact ccgcccagtt ccgcccattc tccgccccat
     9961 ggctgactaa ttttttttat ttatgcagag gccgaggccg cctcggcctc tgagctattc
    10021 cagaagtagt gaggaggctt ttttggaggc ctttcaaata tgtatccgct catgagacaa
    10081 taaccctgat aaatgcttca ataatattga aaaaggaaga gtatgagtat tcaacatttc
    10141 cgtgtcgccc ttattccctt ttttgcggca ttttgccttc ctgtttttgc tcacccagaa
    10201 acgctggtga aagtaaaaga tgctgaagat cagttgggtg cacgagtggg ttacatcgaa
    10261 ctggatctca acagcggtaa gatccttgag agttttcgcc ccgaagaacg ttttccaatg
    10321 atgagcactt ttaaagttct gctatgtggc gcggtattat cccgtattga cgccgggcaa
    10381 gagcaactcg gtcgccgcat acactattct cagaatgact tggttgagta ctcaccagtc
    10441 acagaaaagc atcttacgga tggcatgaca gtaagagaat tatgcagtgc tgccataacc
    10501 atgagtgata acactgcggc caacttactt ctgacaacga tcggaggacc gaaggagcta
    10561 accgcttttt tgcacaacat gggggatcat gtaactcgcc ttgatcgttg ggaaccggag
    10621 ctgaatgaag ccataccaaa cgacgagcgt gacaccacga tgcctgtagc aatggcaaca
    10681 acgttgcgca aactattaac tggcgaacta cttactctag cttcccggca acaattaata
    10741 gactggatgg aggcggataa agttgcagga ccacttctgc gctcggccct tccggctggc
    10801 tggtttattg ctgataaatc tggagccggt gagcgtgggt ctcgcggtat cattgcagca
    10861 ctggggccag atggtaagcc ctcccgtatc gtagttatct acacgacggg gagtcaggca
    10921 actatggatg aacgaaatag acagatcgct gagataggtg cctcactgat taagcattgg
    10981 taactgtcag accaagttta ctcatatata ctttagattg atttaaaact tcatttttaa
    11041 tttaaaagga tctaggtgaa gatccttttt gataatctca tgaccaaaat cccttaacgt
    11101 gagttttcgt tccactgagc gtcagacccc gtagaaaaga tcaaaggatc ttcttgagat
    11161 cctttttttc tgcgcgtaat ctgctgcttg caaacaaaaa aaccaccgct accagcggtg
    11221 gtttgtttgc cggatcaaga gctaccaact ctttttccga aggtaactgg cttcagcaga
    11281 gcgcagatac caaatactgt tcttctagtg tagccgtagt taggccacca cttcaagaac
    11341 tctgtagcac cgcctacata cctcgctctg ctaatcctgt taccagtggc tgctgccagt
    11401 ggcgataagt cgtgtcttac cgggttggac tcaagacgat agttaccgga taaggcgcag
    11461 cggtcgggct gaacgggggg ttcgtgcaca cagcccagct tggagcgaac gacctacacc
    11521 gaactgagat acctacagcg tgagctatga gaaagcgcca cgcttcccga agggagaaag
    11581 gcggacaggt atccggtaag cggcagggtc ggaacaggag agcgcacgag ggagcttcca
    11641 gggggaaacg cctggtatct ttatagtcct gtcgggtttc gccacctctg acttgagcgt
    11701 cgatttttgt gatgctcgtc aggggggcgg agcctatgga aaaacgccag caacgcggcc
    11761 tttttacggt tcctggcctt ttgctggcct tttgctcaca tgttctttcc tgcgttatcc
    11821 cctgattctg tggataaccg tattaccgcc tttgagtgag ctgataccgc tcgccgcagc
    11881 cgaacgaccg agcgcagcga gtcagtgagc gaggaagcgg aagagcgccc aatacgcaaa
    11941 ccgcctctcc ccgcgcgttg gccgattcat taatgcagct ggcacgacag gtttcccgac
    12001 tggaaagcgg gcagtgagcg caacgcaatt aatgtgagtt agctcactca ttaggcaccc
    12061 caggctttac actttatgct tccggctcgt atgttgtgtg gaattgtgag cggataacaa
    12121 tttcacacag gaaacagcta tgaccatgat tacgccaagc gcgcaattaa ccctcactaa
    12181 agggaacaaa agctggagct gcaagcttaa tgtagtctta tgcaatactc ttgtagtctt
    12241 gcaacatggt aacgatgagt tagcaacatg ccttacaagg agagaaaaag caccgtgcat
    12301 gccgattggt ggaagtaagg tggtacgatc gtgccttatt aggaaggcaa cagacgggtc
    12361 tgacatggat tggacgaacc actgaattgc cgcattgcag agatattgta tttaagtgcc
    12421 tagctcgata cataaac
//
