LOCUS       sequence-326381-       12674 bp    DNA     circular SYN 15-DEC-2025
DEFINITION  Doxycycline-inducible Cas9 expression tracked by EGFP fluorescence 
            marker..
ACCESSION   .
VERSION     .
KEYWORDS    .
SOURCE      synthetic DNA construct
  ORGANISM  synthetic DNA construct
REFERENCE   1  (bases 1 to 12674)
  AUTHORS   Chan W, Gottschalk RA, Yao Y, Pomerantz JL, Germain RN
  TITLE     Efficient Immune Cell Genome Engineering with Enhanced CRISPR 
            Editing Tools.
  JOURNAL   Immunohorizons. 2021 Feb 23;5(2):117-132. doi: 
            10.4049/immunohorizons.2000082.
  PUBMED    33622708
REFERENCE   2  (bases 1 to 12674)
  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"; doi: 
            "10.4049/immunohorizons"; journalName: "Immunohorizons"; date: 
            "2021-02-23- 23"; volume: "5"; issue: "2"; pages: "117-132"
COMMENT     Sequence Label: pCW-Cas9-2A-EGFP
FEATURES             Location/Qualifiers
     source          1..12674
                     /mol_type="other DNA"
                     /organism="synthetic DNA construct"
     misc_feature    41..158
                     /label=cPPT/CTS
                     /note="central polypurine tract and central termination 
                     sequence of HIV-1"
     promoter        212..526
                     /label=tight TRE promoter
                     /note="Tet-responsive promoter PTight, consisting of seven 
                     tet operator sequences followed by the minimal CMV 
                     promoter"
     protein_bind    220..238
                     /gene="tetO"
                     /label=tet operator
                     /bound_moiety="tetracycline repressor TetR"
                     /note="bacterial operator O2 for the tetR and tetA genes"
     protein_bind    256..274
                     /gene="tetO"
                     /label=tet operator
                     /bound_moiety="tetracycline repressor TetR"
                     /note="bacterial operator O2 for the tetR and tetA genes"
     protein_bind    291..309
                     /gene="tetO"
                     /label=tet operator
                     /bound_moiety="tetracycline repressor TetR"
                     /note="bacterial operator O2 for the tetR and tetA genes"
     protein_bind    327..345
                     /gene="tetO"
                     /label=tet operator
                     /bound_moiety="tetracycline repressor TetR"
                     /note="bacterial operator O2 for the tetR and tetA genes"
     protein_bind    363..381
                     /gene="tetO"
                     /label=tet operator
                     /bound_moiety="tetracycline repressor TetR"
                     /note="bacterial operator O2 for the tetR and tetA genes"
     protein_bind    398..416
                     /gene="tetO"
                     /label=tet operator
                     /bound_moiety="tetracycline repressor TetR"
                     /note="bacterial operator O2 for the tetR and tetA genes"
     protein_bind    434..452
                     /gene="tetO"
                     /label=tet operator
                     /bound_moiety="tetracycline repressor TetR"
                     /note="bacterial operator O2 for the tetR and tetA genes"
     primer_bind     499..523
                     /label=LNCX
                     /note="Human CMV promoter, forward primer"
     regulatory      543..552
                     /label=Kozak sequence
                     /note="vertebrate consensus sequence for strong initiation 
                     of translation (Kozak, 1987)"
                     /regulatory_class="other"
     CDS             552..617
                     /codon_start=1
                     /product="three tandem FLAG(R) epitope tags, followed by an
                     enterokinase cleavage site"
                     /label=3xFLAG
                     /translation="DYKDHDGDYKDHDIDYKDDDDK"
     CDS             624..644
                     /codon_start=1
                     /product="nuclear localization signal of SV40 (simian virus
                     40) large T antigen"
                     /label=SV40 NLS
                     /translation="PKKKRKV"
     CDS             669..4769
                     /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             669..4769
                     /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             4770..4817
                     /codon_start=1
                     /product="bipartite nuclear localization signal from 
                     nucleoplasmin"
                     /label=nucleoplasmin NLS
                     /translation="KRPAATKKAGQAKKKK"
     CDS             4833..4886
                     /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(4887..4889,4890..5600)
                     /codon_start=1
                     /product="enhanced GFP"
                     /label=EGFP
                     /translation="VSKGEELFTGVVPILVELDGDVNGHKFSVSGEGEGDATYGKLTLK
                     FICTTGKLPVPWPTLVTTLTYGVQCFSRYPDHMKQHDFFKSAMPEGYVQERTIFFKDDG
                     NYKTRAEVKFEGDTLVNRIELKGIDFKEDGNILGHKLEYNYNSHNVYIMADKQKNGIKV
                     NFKIRHNIEDGSVQLADHYQQNTPIGDGPVLLPDNHYLSTQSALSKDPNEKRDHMVLLE
                     FVTAAGITLGMDELYK"
     CDS             join(4887..4889,4890..5600)
                     /codon_start=1
                     /product="the original enhanced GFP (Yang et al., 1996)"
                     /label=EGFP
                     /note="mammalian codon-optimized"
                     /translation="VSKGEELFTGVVPILVELDGDVNGHKFSVSGEGEGDATYGKLTLK
                     FICTTGKLPVPWPTLVTTLTYGVQCFSRYPDHMKQHDFFKSAMPEGYVQERTIFFKDDG
                     NYKTRAEVKFEGDTLVNRIELKGIDFKEDGNILGHKLEYNYNSHNVYIMADKQKNGIKV
                     NFKIRHNIEDGSVQLADHYQQNTPIGDGPVLLPDNHYLSTQSALSKDPNEKRDHMVLLE
                     FVTAAGITLGMDELYK"
     primer_bind     complement(5190..5209)
                     /label=EXFP-R
                     /note="For distinguishing EGFP variants, reverse primer"
     primer_bind     5537..5558
                     /label=EGFP-C
                     /note="EGFP, forward primer"
     promoter        5616..6126
                     /label=hPGK promoter
                     /note="human phosphoglycerate kinase 1 promoter"
     primer_bind     6045..6063
                     /label=hPGK-F
                     /note="Human PGK promoter, forward primer"
     CDS             6136..6732
                     /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(6136..6155)
                     /label=Puro-R
                     /note="Puromycin resistance gene, reverse primer. Also 
                     called puro-variant-R"
     primer_bind     6632..6652
                     /label=Puro-F
                     /note="Puromycin resistance gene, forward primer"
     CDS             6733..6786
                     /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(6787..7404,7405..7410,7411..7446,7447..7449,7450..7485,
                     7486..7488,7489..7524,7525..7533)
                     /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(7573..7593)
                     /label=WPRE-R
                     /note="WPRE, reverse primer"
     CDS             complement(7991..8002)
                     /codon_start=1
                     /product="Factor Xa recognition and cleavage site"
                     /label=Factor Xa site
                     /translation="IEGR"
     LTR             8207..8440
                     /label=3' LTR (Delta-U3)
                     /note="self-inactivating 3' long terminal repeat (LTR) from
                     HIV-1"
     polyA_signal    8512..8633
                     /label=SV40 poly(A) signal
                     /note="SV40 polyadenylation signal"
     primer_bind     complement(8549..8568)
                     /label=SV40pA-R
                     /note="SV40 polyA, reverse primer"
     primer_bind     8603..8622
                     /label=EBV-rev
                     /note="SV40 polyA terminator, reverse primer"
     rep_origin      8673..8808
                     /label=SV40 ori
                     /note="SV40 origin of replication"
     promoter        8808..8879
                     /gene="bla"
                     /label=AmpR promoter
     CDS             join(8880..8948,8949..9740)
                     /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(9098..9117)
                     /label=Amp-R
                     /note="Ampicillin resistance gene, reverse primer"
     rep_origin      9911..10499
                     /direction=RIGHT
                     /label=ori
                     /note="high-copy-number ColE1/pMB1/pBR322/pUC origin of 
                     replication"
     primer_bind     10400..10419
                     /label=pBR322ori-F
                     /note="pBR322 origin, forward primer"
     primer_bind     10653..10670
                     /label=L4440
                     /note="L4440 vector, forward primer"
     protein_bind    10787..10808
                     /label=CAP binding site
                     /bound_moiety="E. coli catabolite activator protein"
                     /note="CAP binding activates transcription in the presence 
                     of cAMP."
     promoter        join(10823..10828,10829..10846,10847..10853)
                     /label=lac promoter
                     /note="promoter for the E. coli lac operon"
     protein_bind    10861..10877
                     /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     10866..10888
                     /label=M13/pUC Reverse
                     /note="In lacZ gene"
     primer_bind     10885..10901
                     /label=M13 rev
                     /note="common sequencing primer, one of multiple similar 
                     variants"
     primer_bind     10885..10901
                     /label=M13 Reverse
                     /note="In lacZ gene. Also called M13-rev"
     primer_bind     10920..10940
                     /label=T3
                     /note="T3 promoter, forward primer"
     promoter        10922..10940
                     /label=T3 promoter
                     /note="promoter for bacteriophage T3 RNA polymerase"
     promoter        10968..11194
                     /label=RSV promoter
                     /note="Rous sarcoma virus enhancer/promoter"
     LTR             11195..11375
                     /label=5' LTR (truncated)
                     /note="truncated 5' long terminal repeat (LTR) from HIV-1"
     misc_feature    11422..11547
                     /label=HIV-1 Psi
                     /note="packaging signal of human immunodeficiency virus 
                     type 1"
     misc_feature    12040..12273
                     /label=RRE
                     /note="The Rev response element (RRE) of HIV-1 allows for 
                     Rev-dependent mRNA export from the nucleus to the 
                     cytoplasm."
     CDS             12458..12502
                     /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"
ORIGIN
        1 aattctcgac ctcgagacaa atggcagtat tcatccacaa ttttaaaaga aaagggggga
       61 ttggggggta cagtgcaggg gaaagaatag tagacataat agcaacagac atacaaacta
      121 aagaattaca aaaacaaatt acaaaaattc aaaattttcg ggtttattac agggacagca
      181 gagatccact ttggccgcga atcgatatgt cgagtttact ccctatcagt gatagagaac
      241 gtatgtcgag tttactccct atcagtgata gagaacgatg tcgagtttac tccctatcag
      301 tgatagagaa cgtatgtcga gtttactccc tatcagtgat agagaacgta tgtcgagttt
      361 actccctatc agtgatagag aacgtatgtc gagtttatcc ctatcagtga tagagaacgt
      421 atgtcgagtt tactccctat cagtgataga gaacgtatgt cgaggtaggc gtgtacggtg
      481 ggaggcctat ataagcagag ctcgtttagt gaaccgtcag atcgcctgga gaattggcta
      541 gcgccaccat ggactataag gaccacgacg gagactacaa ggatcatgat attgattaca
      601 aagacgatga cgataagatg gccccaaaga agaagcggaa ggtcggtatc cacggagtcc
      661 cagcagccga caagaagtac agcatcggcc tggacatcgg caccaactct gtgggctggg
      721 ccgtgatcac cgacgagtac aaggtgccca gcaagaaatt caaggtgctg ggcaacaccg
      781 accggcacag catcaagaag aacctgatcg gagccctgct gttcgacagc ggcgaaacag
      841 ccgaggccac ccggctgaag agaaccgcca gaagaagata caccagacgg aagaaccgga
      901 tctgctatct gcaagagatc ttcagcaacg agatggccaa ggtggacgac agcttcttcc
      961 acagactgga agagtccttc ctggtggaag aggataagaa gcacgagcgg caccccatct
     1021 tcggcaacat cgtggacgag gtggcctacc acgagaagta ccccaccatc taccacctga
     1081 gaaagaaact ggtggacagc accgacaagg ccgacctgcg gctgatctat ctggccctgg
     1141 cccacatgat caagttccgg ggccacttcc tgatcgaggg cgacctgaac cccgacaaca
     1201 gcgacgtgga caagctgttc atccagctgg tgcagaccta caaccagctg ttcgaggaaa
     1261 accccatcaa cgccagcggc gtggacgcca aggccatcct gtctgccaga ctgagcaaga
     1321 gcagacggct ggaaaatctg atcgcccagc tgcccggcga gaagaagaat ggcctgttcg
     1381 gaaacctgat tgccctgagc ctgggcctga cccccaactt caagagcaac ttcgacctgg
     1441 ccgaggatgc caaactgcag ctgagcaagg acacctacga cgacgacctg gacaacctgc
     1501 tggcccagat cggcgaccag tacgccgacc tgtttctggc cgccaagaac ctgtccgacg
     1561 ccatcctgct gagcgacatc ctgagagtga acaccgagat caccaaggcc cccctgagcg
     1621 cctctatgat caagagatac gacgagcacc accaggacct gaccctgctg aaagctctcg
     1681 tgcggcagca gctgcctgag aagtacaaag agattttctt cgaccagagc aagaacggct
     1741 acgccggcta cattgacggc ggagccagcc aggaagagtt ctacaagttc atcaagccca
     1801 tcctggaaaa gatggacggc accgaggaac tgctcgtgaa gctgaacaga gaggacctgc
     1861 tgcggaagca gcggaccttc gacaacggca gcatccccca ccagatccac ctgggagagc
     1921 tgcacgccat tctgcggcgg caggaagatt tttacccatt cctgaaggac aaccgggaaa
     1981 agatcgagaa gatcctgacc ttccgcatcc cctactacgt gggccctctg gccaggggaa
     2041 acagcagatt cgcctggatg accagaaaga gcgaggaaac catcaccccc tggaacttcg
     2101 aggaagtggt ggacaagggc gcttccgccc agagcttcat cgagcggatg accaacttcg
     2161 ataagaacct gcccaacgag aaggtgctgc ccaagcacag cctgctgtac gagtacttca
     2221 ccgtgtataa cgagctgacc aaagtgaaat acgtgaccga gggaatgaga aagcccgcct
     2281 tcctgagcgg cgagcagaaa aaggccatcg tggacctgct gttcaagacc aaccggaaag
     2341 tgaccgtgaa gcagctgaaa gaggactact tcaagaaaat cgagtgcttc gactccgtgg
     2401 aaatctccgg cgtggaagat cggttcaacg cctccctggg cacataccac gatctgctga
     2461 aaattatcaa ggacaaggac ttcctggaca atgaggaaaa cgaggacatt ctggaagata
     2521 tcgtgctgac cctgacactg tttgaggaca gagagatgat cgaggaacgg ctgaaaacct
     2581 atgcccacct gttcgacgac aaagtgatga agcagctgaa gcggcggaga tacaccggct
     2641 ggggcaggct gagccggaag ctgatcaacg gcatccggga caagcagtcc ggcaagacaa
     2701 tcctggattt cctgaagtcc gacggcttcg ccaacagaaa cttcatgcag ctgatccacg
     2761 acgacagcct gacctttaaa gaggacatcc agaaagccca ggtgtccggc cagggcgata
     2821 gcctgcacga gcacattgcc aatctggccg gcagccccgc cattaagaag ggcatcctgc
     2881 agacagtgaa ggtggtggac gagctcgtga aagtgatggg ccggcacaag cccgagaaca
     2941 tcgtgatcga aatggccaga gagaaccaga ccacccagaa gggacagaag aacagccgcg
     3001 agagaatgaa gcggatcgaa gagggcatca aagagctggg cagccagatc ctgaaagaac
     3061 accccgtgga aaacacccag ctgcagaacg agaagctgta cctgtactac ctgcagaatg
     3121 ggcgggatat gtacgtggac caggaactgg acatcaaccg gctgtccgac tacgatgtgg
     3181 accatatcgt gcctcagagc tttctgaagg acgactccat cgacaacaag gtgctgacca
     3241 gaagcgacaa gaaccggggc aagagcgaca acgtgccctc cgaagaggtc gtgaagaaga
     3301 tgaagaacta ctggcggcag ctgctgaacg ccaagctgat tacccagaga aagttcgaca
     3361 atctgaccaa ggccgagaga ggcggcctga gcgaactgga taaggccggc ttcatcaaga
     3421 gacagctggt ggaaacccgg cagatcacaa agcacgtggc acagatcctg gactcccgga
     3481 tgaacactaa gtacgacgag aatgacaagc tgatccggga agtgaaagtg atcaccctga
     3541 agtccaagct ggtgtccgat ttccggaagg atttccagtt ttacaaagtg cgcgagatca
     3601 acaactacca ccacgcccac gacgcctacc tgaacgccgt cgtgggaacc gccctgatca
     3661 aaaagtaccc taagctggaa agcgagttcg tgtacggcga ctacaaggtg tacgacgtgc
     3721 ggaagatgat cgccaagagc gagcaggaaa tcggcaaggc taccgccaag tacttcttct
     3781 acagcaacat catgaacttt ttcaagaccg agattaccct ggccaacggc gagatccgga
     3841 agcggcctct gatcgagaca aacggcgaaa ccggggagat cgtgtgggat aagggccggg
     3901 attttgccac cgtgcggaaa gtgctgagca tgccccaagt gaatatcgtg aaaaagaccg
     3961 aggtgcagac aggcggcttc agcaaagagt ctatcctgcc caagaggaac agcgataagc
     4021 tgatcgccag aaagaaggac tgggacccta agaagtacgg cggcttcgac agccccaccg
     4081 tggcctattc tgtgctggtg gtggccaaag tggaaaaggg caagtccaag aaactgaaga
     4141 gtgtgaaaga gctgctgggg atcaccatca tggaaagaag cagcttcgag aagaatccca
     4201 tcgactttct ggaagccaag ggctacaaag aagtgaaaaa ggacctgatc atcaagctgc
     4261 ctaagtactc cctgttcgag ctggaaaacg gccggaagag aatgctggcc tctgccggcg
     4321 aactgcagaa gggaaacgaa ctggccctgc cctccaaata tgtgaacttc ctgtacctgg
     4381 ccagccacta tgagaagctg aagggctccc ccgaggataa tgagcagaaa cagctgtttg
     4441 tggaacagca caagcactac ctggacgaga tcatcgagca gatcagcgag ttctccaaga
     4501 gagtgatcct ggccgacgct aatctggaca aagtgctgtc cgcctacaac aagcaccggg
     4561 ataagcccat cagagagcag gccgagaata tcatccacct gtttaccctg accaatctgg
     4621 gagcccctgc cgccttcaag tactttgaca ccaccatcga ccggaagagg tacaccagca
     4681 ccaaagaggt gctggacgcc accctgatcc accagagcat caccggcctg tacgagacac
     4741 ggatcgacct gtctcagctg ggaggcgaca aaaggccggc ggccacgaaa aaggccggcc
     4801 aggcaaaaaa gaaaaaggaa ttcggcagtg gagagggcag aggaagtctg ctaacatgcg
     4861 gtgacgtcga ggagaatcct ggcccagtga gcaagggcga ggagctgttc accggggtgg
     4921 tgcccatcct ggtcgagctg gacggcgacg taaacggcca caagttcagc gtgtccggcg
     4981 agggcgaggg cgatgccacc tacggcaagc tgaccctgaa gttcatctgc accaccggca
     5041 agctgcccgt gccctggccc accctcgtga ccaccctgac ctacggcgtg cagtgcttca
     5101 gccgctaccc cgaccacatg aagcagcacg acttcttcaa gtccgccatg cccgaaggct
     5161 acgtccagga gcgcaccatc ttcttcaagg acgacggcaa ctacaagacc cgcgccgagg
     5221 tgaagttcga gggcgacacc ctggtgaacc gcatcgagct gaagggcatc gacttcaagg
     5281 aggacggcaa catcctgggg cacaagctgg agtacaacta caacagccac aacgtctata
     5341 tcatggccga caagcagaag aacggcatca aggtgaactt caagatccgc cacaacatcg
     5401 aggacggcag cgtgcagctc gccgaccact accagcagaa cacccccatc ggcgacggcc
     5461 ccgtgctgct gcccgacaac cactacctga gcacccagtc cgccctgagc aaagacccca
     5521 acgagaagcg cgatcacatg gtcctgctgg agttcgtgac cgccgccggg atcactctcg
     5581 gcatggacga gctgtacaag gaattctaag gatccggggt tggggttgcg ccttttccaa
     5641 ggcagccctg ggtttgcgca gggacgcggc tgctctgggc gtggttccgg gaaacgcagc
     5701 ggcgccgacc ctgggtctcg cacattcttc acgtccgttc gcagcgtcac ccggatcttc
     5761 gccgctaccc ttgtgggccc cccggcgacg cttcctgctc cgcccctaag tcgggaaggt
     5821 tccttgcggt tcgcggcgtg ccggacgtga caaacggaag ccgcacgtct cactagtacc
     5881 ctcgcagacg gacagcgcca gggagcaatg gcagcgcgcc gaccgcgatg ggctgtggcc
     5941 aatagcggct gctcagcagg gcgcgccgag agcagcggcc gggaaggggc ggtgcgggag
     6001 gcggggtgtg gggcggtagt gtgggccctg ttcctgcccg cgcggtgttc cgcattctgc
     6061 aagcctccgg agcgcacgtc ggcagtcggc tccctcgttg accgaatcac cgacctctct
     6121 ccccagcaat tcaccatgac cgagtacaag cccacggtgc gcctcgccac ccgcgacgac
     6181 gtccccaggg ccgtacgcac cctcgccgcc gcgttcgccg actaccccgc cacgcgccac
     6241 accgtcgatc cggaccgcca catcgagcgg gtcaccgagc tgcaagaact cttcctcacg
     6301 cgcgtcgggc tcgacatcgg caaggtgtgg gtcgcggacg acggcgccgc ggtggcggtc
     6361 tggaccacgc cggagagcgt cgaagcgggg gcggtgttcg ccgagatcgg cccgcgcatg
     6421 gccgagttga gcggttcccg gctggccgcg cagcaacaga tggaaggcct cctggcgccg
     6481 caccggccca aggagcccgc gtggttcctg gccaccgtcg gcgtctcgcc cgaccaccag
     6541 ggcaagggtc tgggcagcgc cgtcgtgctc cccggagtgg aggcggccga gcgcgccggg
     6601 gtgcccgcct tcctggagac ctccgcgccc cgcaacctcc ccttctacga gcggctcggc
     6661 ttcaccgtca ccgccgacgt cgaggtgccc gaaggaccgc gcacctggtg catgacccgc
     6721 aagcccggtg ccgaaggtag aggttctctc ctcacttgtg gtgatgttga agaaaaccct
     6781 ggtccaatgt ctagactgga caagagcaaa gtcataaacg gagctctgga attactcaat
     6841 ggtgtcggta tcgaaggcct gacgacaagg aaactcgctc aaaagctggg agttgagcag
     6901 cctaccctgt actggcacgt gaagaacaag cgggccctgc tcgatgccct gccaatcgag
     6961 atgctggaca ggcatcatac ccacttctgc cccctggaag gcgagtcatg gcaagacttt
     7021 ctgcggaaca acgccaagtc ataccgctgt gctctcctct cacatcgcga cggggctaaa
     7081 gtgcatctcg gcacccgccc aacagagaaa cagtacgaaa ccctggaaaa tcagctcgcg
     7141 ttcctgtgtc agcaaggctt ctccctggag aacgcactgt acgctctgtc cgccgtgggc
     7201 cactttacac tgggctgcgt attggaggaa caggagcatc aagtagcaaa agaggaaaga
     7261 gagacaccta ccaccgattc tatgccccca cttctgagac aagcaattga gctgttcgac
     7321 cggcagggag ccgaacctgc cttccttttc ggcctggaac taatcatatg tggcctggag
     7381 aaacagctaa agtgcgaaag cggcgggccg accgacgccc ttgacgattt tgacttagac
     7441 atgctcccag ccgatgccct tgacgacttt gaccttgata tgctgcctgc tgacgctctt
     7501 gacgattttg accttgacat gctccccggg taactaagta aggatcgatc caagatatcg
     7561 tattcttaac tatgttgctc cttttacgct atgtggatac gctgctttaa tgcctttgta
     7621 tcatgctatt gcttcccgta tggctttcat tttctcctcc ttgtataaat cctggttgct
     7681 gtctctttat gaggagttgt ggcccgttgt caggcaacgt ggcgtggtgt gcactgtgtt
     7741 tgctgacgca acccccactg gttggggcat tgccaccacc tgtcagctcc tttccgggac
     7801 tttcgctttc cccctcccta ttgccacggc ggaactcatc gccgcctgcc ttgcccgctg
     7861 ctggacaggg gctcggctgt tgggcactga caattccgtg gtgttgtcgg ggaagctgac
     7921 gtcctttcca tggctgctcg cctgtgttgc cacctggatt ctgcgcggga cgtccttctg
     7981 ctacgtccct tcggccctca atccagcgga ccttccttcc cgcggcctgc tgccggctct
     8041 gcggcctctt ccgcgtcttc gccttcgccc tcagacgagt cggatctccc tttgggccgc
     8101 ctccccgcct gtttcgcctc ggcgtccgga ctagaggtac ctttaagacc aatgacttac
     8161 aaggcagctg tagatcttag ccacttttta aaagaaaagg ggggactgga agggctaatt
     8221 cactcccaac gaagacaaga tctgcttttt gcttgtactg ggtctctctg gttagaccag
     8281 atctgagcct gggagctctc tggctaacta gggaacccac tgcttaagcc tcaataaagc
     8341 ttgccttgag tgcttcaagt agtgtgtgcc cgtctgttgt gtgactctgg taactagaga
     8401 tccctcagac ccttttagtc agtgtggaaa atctctagca gtagtagttc atgtcatctt
     8461 attattcagt atttataact tgcaaagaaa tgaatatcag agagtgagag gaacttgttt
     8521 attgcagctt ataatggtta caaataaagc aatagcatca caaatttcac aaataaagca
     8581 tttttttcac tgcattctag ttgtggtttg tccaaactca tcaatgtatc ttatcatgtc
     8641 tggctctagc tatcccgccc ctaactccgc ccatcccgcc cctaactccg cccagttccg
     8701 cccattctcc gccccatggc tgactaattt tttttattta tgcagaggcc gaggccgcct
     8761 cggcctctga gctattccag aagtagtgag gaggcttttt tggaggcctt tcaaatatgt
     8821 atccgctcat gagacaataa ccctgataaa tgcttcaata atattgaaaa aggaagagta
     8881 tgagtattca acatttccgt gtcgccctta ttcccttttt tgcggcattt tgccttcctg
     8941 tttttgctca cccagaaacg ctggtgaaag taaaagatgc tgaagatcag ttgggtgcac
     9001 gagtgggtta catcgaactg gatctcaaca gcggtaagat ccttgagagt tttcgccccg
     9061 aagaacgttt tccaatgatg agcactttta aagttctgct atgtggcgcg gtattatccc
     9121 gtattgacgc cgggcaagag caactcggtc gccgcataca ctattctcag aatgacttgg
     9181 ttgagtactc accagtcaca gaaaagcatc ttacggatgg catgacagta agagaattat
     9241 gcagtgctgc cataaccatg agtgataaca ctgcggccaa cttacttctg acaacgatcg
     9301 gaggaccgaa ggagctaacc gcttttttgc acaacatggg ggatcatgta actcgccttg
     9361 atcgttggga accggagctg aatgaagcca taccaaacga cgagcgtgac accacgatgc
     9421 ctgtagcaat ggcaacaacg ttgcgcaaac tattaactgg cgaactactt actctagctt
     9481 cccggcaaca attaatagac tggatggagg cggataaagt tgcaggacca cttctgcgct
     9541 cggcccttcc ggctggctgg tttattgctg ataaatctgg agccggtgag cgtgggtctc
     9601 gcggtatcat tgcagcactg gggccagatg gtaagccctc ccgtatcgta gttatctaca
     9661 cgacggggag tcaggcaact atggatgaac gaaatagaca gatcgctgag ataggtgcct
     9721 cactgattaa gcattggtaa ctgtcagacc aagtttactc atatatactt tagattgatt
     9781 taaaacttca tttttaattt aaaaggatct aggtgaagat cctttttgat aatctcatga
     9841 ccaaaatccc ttaacgtgag ttttcgttcc actgagcgtc agaccccgta gaaaagatca
     9901 aaggatcttc ttgagatcct ttttttctgc gcgtaatctg ctgcttgcaa acaaaaaaac
     9961 caccgctacc agcggtggtt tgtttgccgg atcaagagct accaactctt tttccgaagg
    10021 taactggctt cagcagagcg cagataccaa atactgttct tctagtgtag ccgtagttag
    10081 gccaccactt caagaactct gtagcaccgc ctacatacct cgctctgcta atcctgttac
    10141 cagtggctgc tgccagtggc gataagtcgt gtcttaccgg gttggactca agacgatagt
    10201 taccggataa ggcgcagcgg tcgggctgaa cggggggttc gtgcacacag cccagcttgg
    10261 agcgaacgac ctacaccgaa ctgagatacc tacagcgtga gctatgagaa agcgccacgc
    10321 ttcccgaagg gagaaaggcg gacaggtatc cggtaagcgg cagggtcgga acaggagagc
    10381 gcacgaggga gcttccaggg ggaaacgcct ggtatcttta tagtcctgtc gggtttcgcc
    10441 acctctgact tgagcgtcga tttttgtgat gctcgtcagg ggggcggagc ctatggaaaa
    10501 acgccagcaa cgcggccttt ttacggttcc tggccttttg ctggcctttt gctcacatgt
    10561 tctttcctgc gttatcccct gattctgtgg ataaccgtat taccgccttt gagtgagctg
    10621 ataccgctcg ccgcagccga acgaccgagc gcagcgagtc agtgagcgag gaagcggaag
    10681 agcgcccaat acgcaaaccg cctctccccg cgcgttggcc gattcattaa tgcagctggc
    10741 acgacaggtt tcccgactgg aaagcgggca gtgagcgcaa cgcaattaat gtgagttagc
    10801 tcactcatta ggcaccccag gctttacact ttatgcttcc ggctcgtatg ttgtgtggaa
    10861 ttgtgagcgg ataacaattt cacacaggaa acagctatga ccatgattac gccaagcgcg
    10921 caattaaccc tcactaaagg gaacaaaagc tggagctgca agcttaatgt agtcttatgc
    10981 aatactcttg tagtcttgca acatggtaac gatgagttag caacatgcct tacaaggaga
    11041 gaaaaagcac cgtgcatgcc gattggtgga agtaaggtgg tacgatcgtg ccttattagg
    11101 aaggcaacag acgggtctga catggattgg acgaaccact gaattgccgc attgcagaga
    11161 tattgtattt aagtgcctag ctcgatacat aaacgggtct ctctggttag accagatctg
    11221 agcctgggag ctctctggct aactagggaa cccactgctt aagcctcaat aaagcttgcc
    11281 ttgagtgctt caagtagtgt gtgcccgtct gttgtgtgac tctggtaact agagatccct
    11341 cagacccttt tagtcagtgt ggaaaatctc tagcagtggc gcccgaacag ggacttgaaa
    11401 gcgaaaggga aaccagagga gctctctcga cgcaggactc ggcttgctga agcgcgcacg
    11461 gcaagaggcg aggggcggcg actggtgagt acgccaaaaa ttttgactag cggaggctag
    11521 aaggagagag atgggtgcga gagcgtcagt attaagcggg ggagaattag atcgcgatgg
    11581 gaaaaaattc ggttaaggcc agggggaaag aaaaaatata aattaaaaca tatagtatgg
    11641 gcaagcaggg agctagaacg attcgcagtt aatcctggcc tgttagaaac atcagaaggc
    11701 tgtagacaaa tactgggaca gctacaacca tcccttcaga caggatcaga agaacttaga
    11761 tcattatata atacagtagc aaccctctat tgtgtgcatc aaaggataga gataaaagac
    11821 accaaggaag ctttagacaa gatagaggaa gagcaaaaca aaagtaagac caccgcacag
    11881 caagcggccg ctgatcttca gacctggagg aggagatatg agggacaatt ggagaagtga
    11941 attatataaa tataaagtag taaaaattga accattagga gtagcaccca ccaaggcaaa
    12001 gagaagagtg gtgcagagag aaaaaagagc agtgggaata ggagctttgt tccttgggtt
    12061 cttgggagca gcaggaagca ctatgggcgc agcgtcaatg acgctgacgg tacaggccag
    12121 acaattattg tctggtatag tgcagcagca gaacaatttg ctgagggcta ttgaggcgca
    12181 acagcatctg ttgcaactca cagtctgggg catcaagcag ctccaggcaa gaatcctggc
    12241 tgtggaaaga tacctaaagg atcaacagct cctggggatt tggggttgct ctggaaaact
    12301 catttgcacc actgctgtgc cttggaatgc tagttggagt aataaatctc tggaacagat
    12361 ttggaatcac acgacctgga tggagtggga cagagaaatt aacaattaca caagcttaat
    12421 acactcctta attgaagaat cgcaaaacca gcaagaaaag aatgaacaag aattattgga
    12481 attagataaa tgggcaagtt tgtggaattg gtttaacata acaaattggc tgtggtatat
    12541 aaaattattc ataatgatag taggaggctt ggtaggttta agaatagttt ttgctgtact
    12601 ttctatagtg aatagagtta ggcagggata ttcaccatta tcgtttcaga cccacctccc
    12661 aaccccgagg ggac
//
