LOCUS       sequence-338370-       12576 bp    DNA     circular SYN 15-DEC-2025
DEFINITION  All-in-one doxycycline inducible lentiviral vector for expression of
            Cas9 in combination with turbo GFP using the P2A self-cleaving 
            peptide.
ACCESSION   .
VERSION     .
KEYWORDS    .
SOURCE      synthetic DNA construct
  ORGANISM  synthetic DNA construct
REFERENCE   1  (bases 1 to 12576)
  AUTHORS   Niu Y, Ferreira Azevedo CA, Li X, Kamali E, Haagen Nielsen O, 
            Storgaard Sorensen C, Frodin M
  TITLE     Multiparametric and accurate functional analysis of genetic sequence
            variants using CRISPR-Select.
  JOURNAL   Nat Genet. 2022 Dec 5. doi: 10.1038/s41588-022-01224-7.
  PUBMED    36471068
REFERENCE   2  (bases 1 to 12576)
  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: "Nat Genet. 
            2022 Dec 5. doi: 10.1038/s41588-022-01224-7."
COMMENT     Sequence Label: pCW57-GFP-2A-Cas9
FEATURES             Location/Qualifiers
     source          1..12576
                     /mol_type="other DNA"
                     /organism="synthetic DNA construct"
     protein_bind    1..19
                     /gene="tetO"
                     /label=tet operator
                     /bound_moiety="tetracycline repressor TetR"
                     /note="bacterial operator O2 for the tetR and tetA genes"
     protein_bind    37..55
                     /gene="tetO"
                     /label=tet operator
                     /bound_moiety="tetracycline repressor TetR"
                     /note="bacterial operator O2 for the tetR and tetA genes"
     protein_bind    72..90
                     /gene="tetO"
                     /label=tet operator
                     /bound_moiety="tetracycline repressor TetR"
                     /note="bacterial operator O2 for the tetR and tetA genes"
     protein_bind    108..126
                     /gene="tetO"
                     /label=tet operator
                     /bound_moiety="tetracycline repressor TetR"
                     /note="bacterial operator O2 for the tetR and tetA genes"
     protein_bind    144..162
                     /gene="tetO"
                     /label=tet operator
                     /bound_moiety="tetracycline repressor TetR"
                     /note="bacterial operator O2 for the tetR and tetA genes"
     protein_bind    179..197
                     /gene="tetO"
                     /label=tet operator
                     /bound_moiety="tetracycline repressor TetR"
                     /note="bacterial operator O2 for the tetR and tetA genes"
     protein_bind    215..233
                     /gene="tetO"
                     /label=tet operator
                     /bound_moiety="tetracycline repressor TetR"
                     /note="bacterial operator O2 for the tetR and tetA genes"
     primer_bind     280..304
                     /label=LNCX
                     /note="Human CMV promoter, forward primer"
     regulatory      330..339
                     /label=Kozak sequence
                     /note="vertebrate consensus sequence for strong initiation 
                     of translation (Kozak, 1987)"
                     /regulatory_class="other"
     CDS             336..1031
                     /codon_start=1
                     /product="improved green fluorescent protein from 
                     Pontellina plumata (Evdokimov et al., 2006)"
                     /label=TurboGFP
                     /note="mammalian codon-optimized"
                     /translation="MESDESGLPAMEIECRITGTLNGVEFELVGGGEGTPEQGRMTNKM
                     KSTKGALTFSPYLLSHVMGYGFYHFGTYPSGYENPFLHAINNGGYTNTRIEKYEDGGVL
                     HVSFSYRYEAGRVIGDFKVMGTGFPEDSVIFTDKIIRSNATVEHLHPMGDNDLDGSFTR
                     TFSLRDGGYYSSVVDSHMHFKSAIHPSILQNGGPMFAFRRVEEDHSNTELGIVEYQHAF
                     KTPDADAGEE"
     CDS             1044..1100
                     /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             1101..1121
                     /codon_start=1
                     /product="nuclear localization signal of SV40 (simian virus
                     40) large T antigen"
                     /label=SV40 NLS
                     /translation="PKKKRKV"
     CDS             1146..5246
                     /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             1146..5246
                     /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             5247..5294
                     /codon_start=1
                     /product="bipartite nuclear localization signal from 
                     nucleoplasmin"
                     /label=nucleoplasmin NLS
                     /translation="KRPAATKKAGQAKKKK"
     promoter        5299..5809
                     /label=hPGK promoter
                     /note="human phosphoglycerate kinase 1 promoter"
     primer_bind     5728..5746
                     /label=hPGK-F
                     /note="Human PGK promoter, forward primer"
     CDS             5819..6415
                     /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(5819..5838)
                     /label=Puro-R
                     /note="Puromycin resistance gene, reverse primer. Also 
                     called puro-variant-R"
     primer_bind     6315..6335
                     /label=Puro-F
                     /note="Puromycin resistance gene, forward primer"
     CDS             6416..6469
                     /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(6470..7087,7088..7093,7094..7129,7130..7132,7133..7168,
                     7169..7171,7172..7207,7208..7216)
                     /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(7256..7276)
                     /label=WPRE-R
                     /note="WPRE, reverse primer"
     CDS             complement(7674..7685)
                     /codon_start=1
                     /product="Factor Xa recognition and cleavage site"
                     /label=Factor Xa site
                     /translation="IEGR"
     LTR             7890..8123
                     /label=3' LTR (Delta-U3)
                     /note="self-inactivating 3' long terminal repeat (LTR) from
                     HIV-1"
     polyA_signal    8195..8316
                     /label=SV40 poly(A) signal
                     /note="SV40 polyadenylation signal"
     primer_bind     complement(8232..8251)
                     /label=SV40pA-R
                     /note="SV40 polyA, reverse primer"
     primer_bind     8286..8305
                     /label=EBV-rev
                     /note="SV40 polyA terminator, reverse primer"
     rep_origin      8356..8491
                     /label=SV40 ori
                     /note="SV40 origin of replication"
     promoter        8491..8562
                     /gene="bla"
                     /label=AmpR promoter
     CDS             join(8563..8631,8632..9423)
                     /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(8781..8800)
                     /label=Amp-R
                     /note="Ampicillin resistance gene, reverse primer"
     rep_origin      9594..10182
                     /direction=RIGHT
                     /label=ori
                     /note="high-copy-number ColE1/pMB1/pBR322/pUC origin of 
                     replication"
     primer_bind     10083..10102
                     /label=pBR322ori-F
                     /note="pBR322 origin, forward primer"
     primer_bind     10336..10353
                     /label=L4440
                     /note="L4440 vector, forward primer"
     protein_bind    10470..10491
                     /label=CAP binding site
                     /bound_moiety="E. coli catabolite activator protein"
                     /note="CAP binding activates transcription in the presence 
                     of cAMP."
     promoter        join(10506..10511,10512..10529,10530..10536)
                     /label=lac promoter
                     /note="promoter for the E. coli lac operon"
     protein_bind    10544..10560
                     /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     10549..10571
                     /label=M13/pUC Reverse
                     /note="In lacZ gene"
     primer_bind     10568..10584
                     /label=M13 rev
                     /note="common sequencing primer, one of multiple similar 
                     variants"
     primer_bind     10568..10584
                     /label=M13 Reverse
                     /note="In lacZ gene. Also called M13-rev"
     primer_bind     10603..10623
                     /label=T3
                     /note="T3 promoter, forward primer"
     promoter        10605..10623
                     /label=T3 promoter
                     /note="promoter for bacteriophage T3 RNA polymerase"
     promoter        10651..10877
                     /label=RSV promoter
                     /note="Rous sarcoma virus enhancer/promoter"
     LTR             10878..11058
                     /label=5' LTR (truncated)
                     /note="truncated 5' long terminal repeat (LTR) from HIV-1"
     misc_feature    11105..11230
                     /label=HIV-1 Psi
                     /note="packaging signal of human immunodeficiency virus 
                     type 1"
     misc_feature    11723..11956
                     /label=RRE
                     /note="The Rev response element (RRE) of HIV-1 allows for 
                     Rev-dependent mRNA export from the nucleus to the 
                     cytoplasm."
     CDS             12141..12185
                     /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    12398..12515
                     /label=cPPT/CTS
                     /note="central polypurine tract and central termination 
                     sequence of HIV-1"
     promoter        join(12569..12576,1..307)
                     /label=tight TRE promoter
                     /note="Tet-responsive promoter PTight, consisting of seven 
                     tet operator sequences followed by the minimal CMV 
                     promoter"
ORIGIN
        1 tccctatcag tgatagagaa cgtatgtcga gtttactccc tatcagtgat agagaacgat
       61 gtcgagttta ctccctatca gtgatagaga acgtatgtcg agtttactcc ctatcagtga
      121 tagagaacgt atgtcgagtt tactccctat cagtgataga gaacgtatgt cgagtttatc
      181 cctatcagtg atagagaacg tatgtcgagt ttactcccta tcagtgatag agaacgtatg
      241 tcgaggtagg cgtgtacggt gggaggccta tataagcaga gctcgtttag tgaaccgtca
      301 gatcgcctgg agaattggct agcgaattcg ccaccatgga gagcgacgag agcggcctgc
      361 ccgccatgga gatcgagtgc cgcatcaccg gcaccctgaa cggcgtggag ttcgagctgg
      421 tgggcggcgg agagggcacc cccgagcagg gccgcatgac caacaagatg aagagcacca
      481 aaggcgccct gaccttcagc ccctacctgc tgagccacgt gatgggctac ggcttctacc
      541 acttcggcac ctaccccagc ggctacgaga accccttcct gcacgccatc aacaacggcg
      601 gctacaccaa cacccgcatc gagaagtacg aggacggcgg cgtgctgcac gtgagcttca
      661 gctaccgcta cgaggccggc cgcgtgatcg gcgacttcaa ggtgatgggc accggcttcc
      721 ccgaggacag cgtgatcttc accgacaaga tcatccgcag caacgccacc gtggagcacc
      781 tgcaccccat gggcgataac gatctggatg gcagcttcac ccgcaccttc agcctgcgcg
      841 acggcggcta ctacagctcc gtggtggaca gccacatgca cttcaagagc gccatccacc
      901 ccagcatcct gcagaacggg ggccccatgt tcgccttccg ccgcgtggag gaggatcaca
      961 gcaacaccga gctgggcatc gtggagtacc agcacgcctt caagaccccg gatgcagatg
     1021 ccggtgaaga aaccggttcc ggagccacga acttctctct gttaaagcaa gcaggagacg
     1081 tggaagaaaa ccccggtcct ccaaagaaga agcggaaggt cggtatccac ggagtcccag
     1141 cagccgacaa gaagtacagc atcggcctgg acatcggcac caactctgtg ggctgggccg
     1201 tgatcaccga cgagtacaag gtgcccagca agaaattcaa ggtgctgggc aacaccgacc
     1261 ggcacagcat caagaagaac ctgatcggag ccctgctgtt cgacagcggc gaaacagccg
     1321 aggccacccg gctgaagaga accgccagaa gaagatacac cagacggaag aaccggatct
     1381 gctatctgca agagatcttc agcaacgaga tggccaaggt ggacgacagc ttcttccaca
     1441 gactggaaga gtccttcctg gtggaagagg ataagaagca cgagcggcac cccatcttcg
     1501 gcaacatcgt ggacgaggtg gcctaccacg agaagtaccc caccatctac cacctgagaa
     1561 agaaactggt ggacagcacc gacaaggccg acctgcggct gatctatctg gccctggccc
     1621 acatgatcaa gttccggggc cacttcctga tcgagggcga cctgaacccc gacaacagcg
     1681 acgtggacaa gctgttcatc cagctggtgc agacctacaa ccagctgttc gaggaaaacc
     1741 ccatcaacgc cagcggcgtg gacgccaagg ccatcctgtc tgccagactg agcaagagca
     1801 gacggctgga aaatctgatc gcccagctgc ccggcgagaa gaagaatggc ctgttcggaa
     1861 acctgattgc cctgagcctg ggcctgaccc ccaacttcaa gagcaacttc gacctggccg
     1921 aggatgccaa actgcagctg agcaaggaca cctacgacga cgacctggac aacctgctgg
     1981 cccagatcgg cgaccagtac gccgacctgt ttctggccgc caagaacctg tccgacgcca
     2041 tcctgctgag cgacatcctg agagtgaaca ccgagatcac caaggccccc ctgagcgcct
     2101 ctatgatcaa gagatacgac gagcaccacc aggacctgac cctgctgaaa gctctcgtgc
     2161 ggcagcagct gcctgagaag tacaaagaga ttttcttcga ccagagcaag aacggctacg
     2221 ccggctacat tgacggcgga gccagccagg aagagttcta caagttcatc aagcccatcc
     2281 tggaaaagat ggacggcacc gaggaactgc tcgtgaagct gaacagagag gacctgctgc
     2341 ggaagcagcg gaccttcgac aacggcagca tcccccacca gatccacctg ggagagctgc
     2401 acgccattct gcggcggcag gaagattttt acccattcct gaaggacaac cgggaaaaga
     2461 tcgagaagat cctgaccttc cgcatcccct actacgtggg ccctctggcc aggggaaaca
     2521 gcagattcgc ctggatgacc agaaagagcg aggaaaccat caccccctgg aacttcgagg
     2581 aagtggtgga caagggcgct tccgcccaga gcttcatcga gcggatgacc aacttcgata
     2641 agaacctgcc caacgagaag gtgctgccca agcacagcct gctgtacgag tacttcaccg
     2701 tgtataacga gctgaccaaa gtgaaatacg tgaccgaggg aatgagaaag cccgccttcc
     2761 tgagcggcga gcagaaaaag gccatcgtgg acctgctgtt caagaccaac cggaaagtga
     2821 ccgtgaagca gctgaaagag gactacttca agaaaatcga gtgcttcgac tccgtggaaa
     2881 tctccggcgt ggaagatcgg ttcaacgcct ccctgggcac ataccacgat ctgctgaaaa
     2941 ttatcaagga caaggacttc ctggacaatg aggaaaacga ggacattctg gaagatatcg
     3001 tgctgaccct gacactgttt gaggacagag agatgatcga ggaacggctg aaaacctatg
     3061 cccacctgtt cgacgacaaa gtgatgaagc agctgaagcg gcggagatac accggctggg
     3121 gcaggctgag ccggaagctg atcaacggca tccgggacaa gcagtccggc aagacaatcc
     3181 tggatttcct gaagtccgac ggcttcgcca acagaaactt catgcagctg atccacgacg
     3241 acagcctgac ctttaaagag gacatccaga aagcccaggt gtccggccag ggcgatagcc
     3301 tgcacgagca cattgccaat ctggccggca gccccgccat taagaagggc atcctgcaga
     3361 cagtgaaggt ggtggacgag ctcgtgaaag tgatgggccg gcacaagccc gagaacatcg
     3421 tgatcgaaat ggccagagag aaccagacca cccagaaggg acagaagaac agccgcgaga
     3481 gaatgaagcg gatcgaagag ggcatcaaag agctgggcag ccagatcctg aaagaacacc
     3541 ccgtggaaaa cacccagctg cagaacgaga agctgtacct gtactacctg cagaatgggc
     3601 gggatatgta cgtggaccag gaactggaca tcaaccggct gtccgactac gatgtggacc
     3661 atatcgtgcc tcagagcttt ctgaaggacg actccatcga caacaaggtg ctgaccagaa
     3721 gcgacaagaa ccggggcaag agcgacaacg tgccctccga agaggtcgtg aagaagatga
     3781 agaactactg gcggcagctg ctgaacgcca agctgattac ccagagaaag ttcgacaatc
     3841 tgaccaaggc cgagagaggc ggcctgagcg aactggataa ggccggcttc atcaagagac
     3901 agctggtgga aacccggcag atcacaaagc acgtggcaca gatcctggac tcccggatga
     3961 acactaagta cgacgagaat gacaagctga tccgggaagt gaaagtgatc accctgaagt
     4021 ccaagctggt gtccgatttc cggaaggatt tccagtttta caaagtgcgc gagatcaaca
     4081 actaccacca cgcccacgac gcctacctga acgccgtcgt gggaaccgcc ctgatcaaaa
     4141 agtaccctaa gctggaaagc gagttcgtgt acggcgacta caaggtgtac gacgtgcgga
     4201 agatgatcgc caagagcgag caggaaatcg gcaaggctac cgccaagtac ttcttctaca
     4261 gcaacatcat gaactttttc aagaccgaga ttaccctggc caacggcgag atccggaagc
     4321 ggcctctgat cgagacaaac ggcgaaaccg gggagatcgt gtgggataag ggccgggatt
     4381 ttgccaccgt gcggaaagtg ctgagcatgc cccaagtgaa tatcgtgaaa aagaccgagg
     4441 tgcagacagg cggcttcagc aaagagtcta tcctgcccaa gaggaacagc gataagctga
     4501 tcgccagaaa gaaggactgg gaccctaaga agtacggcgg cttcgacagc cccaccgtgg
     4561 cctattctgt gctggtggtg gccaaagtgg aaaagggcaa gtccaagaaa ctgaagagtg
     4621 tgaaagagct gctggggatc accatcatgg aaagaagcag cttcgagaag aatcccatcg
     4681 actttctgga agccaagggc tacaaagaag tgaaaaagga cctgatcatc aagctgccta
     4741 agtactccct gttcgagctg gaaaacggcc ggaagagaat gctggcctct gccggcgaac
     4801 tgcagaaggg aaacgaactg gccctgccct ccaaatatgt gaacttcctg tacctggcca
     4861 gccactatga gaagctgaag ggctcccccg aggataatga gcagaaacag ctgtttgtgg
     4921 aacagcacaa gcactacctg gacgagatca tcgagcagat cagcgagttc tccaagagag
     4981 tgatcctggc cgacgctaat ctggacaaag tgctgtccgc ctacaacaag caccgggata
     5041 agcccatcag agagcaggcc gagaatatca tccacctgtt taccctgacc aatctgggag
     5101 cccctgccgc cttcaagtac tttgacacca ccatcgaccg gaagaggtac accagcacca
     5161 aagaggtgct ggacgccacc ctgatccacc agagcatcac cggcctgtac gagacacgga
     5221 tcgacctgtc tcagctggga ggcgacaaaa ggccggcggc cacgaaaaag gccggccagg
     5281 caaaaaagaa aaagtaacgg ggttggggtt gcgccttttc caaggcagcc ctgggtttgc
     5341 gcagggacgc ggctgctctg ggcgtggttc cgggaaacgc agcggcgccg accctgggtc
     5401 tcgcacattc ttcacgtccg ttcgcagcgt cacccggatc ttcgccgcta cccttgtggg
     5461 ccccccggcg acgcttcctg ctccgcccct aagtcgggaa ggttccttgc ggttcgcggc
     5521 gtgccggacg tgacaaacgg aagccgcacg tctcactagt accctcgcag acggacagcg
     5581 ccagggagca atggcagcgc gccgaccgcg atgggctgtg gccaatagcg gctgctcagc
     5641 agggcgcgcc gagagcagcg gccgggaagg ggcggtgcgg gaggcggggt gtggggcggt
     5701 agtgtgggcc ctgttcctgc ccgcgcggtg ttccgcattc tgcaagcctc cggagcgcac
     5761 gtcggcagtc ggctccctcg ttgaccgaat caccgacctc tctccccagc aattcaccat
     5821 gaccgagtac aagcccacgg tgcgcctcgc cacccgcgac gacgtcccca gggccgtacg
     5881 caccctcgcc gccgcgttcg ccgactaccc cgccacgcgc cacaccgtcg atccggaccg
     5941 ccacatcgag cgggtcaccg agctgcaaga actcttcctc acgcgcgtcg ggctcgacat
     6001 cggcaaggtg tgggtcgcgg acgacggcgc cgcggtggcg gtctggacca cgccggagag
     6061 cgtcgaagcg ggggcggtgt tcgccgagat cggcccgcgc atggccgagt tgagcggttc
     6121 ccggctggcc gcgcagcaac agatggaagg cctcctggcg ccgcaccggc ccaaggagcc
     6181 cgcgtggttc ctggccaccg tcggcgtctc gcccgaccac cagggcaagg gtctgggcag
     6241 cgccgtcgtg ctccccggag tggaggcggc cgagcgcgcc ggggtgcccg ccttcctgga
     6301 gacctccgcg ccccgcaacc tccccttcta cgagcggctc ggcttcaccg tcaccgccga
     6361 cgtcgaggtg cccgaaggac cgcgcacctg gtgcatgacc cgcaagcccg gtgccgaagg
     6421 tagaggttct ctcctcactt gtggtgatgt tgaagaaaac cctggtccaa tgtctagact
     6481 ggacaagagc aaagtcataa acggagctct ggaattactc aatggtgtcg gtatcgaagg
     6541 cctgacgaca aggaaactcg ctcaaaagct gggagttgag cagcctaccc tgtactggca
     6601 cgtgaagaac aagcgggccc tgctcgatgc cctgccaatc gagatgctgg acaggcatca
     6661 tacccacttc tgccccctgg aaggcgagtc atggcaagac tttctgcgga acaacgccaa
     6721 gtcataccgc tgtgctctcc tctcacatcg cgacggggct aaagtgcatc tcggcacccg
     6781 cccaacagag aaacagtacg aaaccctgga aaatcagctc gcgttcctgt gtcagcaagg
     6841 cttctccctg gagaacgcac tgtacgctct gtccgccgtg ggccacttta cactgggctg
     6901 cgtattggag gaacaggagc atcaagtagc aaaagaggaa agagagacac ctaccaccga
     6961 ttctatgccc ccacttctga gacaagcaat tgagctgttc gaccggcagg gagccgaacc
     7021 tgccttcctt ttcggcctgg aactaatcat atgtggcctg gagaaacagc taaagtgcga
     7081 aagcggcggg ccgaccgacg cccttgacga ttttgactta gacatgctcc cagccgatgc
     7141 ccttgacgac tttgaccttg atatgctgcc tgctgacgct cttgacgatt ttgaccttga
     7201 catgctcccc gggtaactaa gtaaggatcg atccaagata tcgtattctt aactatgttg
     7261 ctccttttac gctatgtgga tacgctgctt taatgccttt gtatcatgct attgcttccc
     7321 gtatggcttt cattttctcc tccttgtata aatcctggtt gctgtctctt tatgaggagt
     7381 tgtggcccgt tgtcaggcaa cgtggcgtgg tgtgcactgt gtttgctgac gcaaccccca
     7441 ctggttgggg cattgccacc acctgtcagc tcctttccgg gactttcgct ttccccctcc
     7501 ctattgccac ggcggaactc atcgccgcct gccttgcccg ctgctggaca ggggctcggc
     7561 tgttgggcac tgacaattcc gtggtgttgt cggggaagct gacgtccttt ccatggctgc
     7621 tcgcctgtgt tgccacctgg attctgcgcg ggacgtcctt ctgctacgtc ccttcggccc
     7681 tcaatccagc ggaccttcct tcccgcggcc tgctgccggc tctgcggcct cttccgcgtc
     7741 ttcgccttcg ccctcagacg agtcggatct ccctttgggc cgcctccccg cctgtttcgc
     7801 ctcggcgtcc ggactagagg tacctttaag accaatgact tacaaggcag ctgtagatct
     7861 tagccacttt ttaaaagaaa aggggggact ggaagggcta attcactccc aacgaagaca
     7921 agatctgctt tttgcttgta ctgggtctct ctggttagac cagatctgag cctgggagct
     7981 ctctggctaa ctagggaacc cactgcttaa gcctcaataa agcttgcctt gagtgcttca
     8041 agtagtgtgt gcccgtctgt tgtgtgactc tggtaactag agatccctca gaccctttta
     8101 gtcagtgtgg aaaatctcta gcagtagtag ttcatgtcat cttattattc agtatttata
     8161 acttgcaaag aaatgaatat cagagagtga gaggaacttg tttattgcag cttataatgg
     8221 ttacaaataa agcaatagca tcacaaattt cacaaataaa gcattttttt cactgcattc
     8281 tagttgtggt ttgtccaaac tcatcaatgt atcttatcat gtctggctct agctatcccg
     8341 cccctaactc cgcccatccc gcccctaact ccgcccagtt ccgcccattc tccgccccat
     8401 ggctgactaa ttttttttat ttatgcagag gccgaggccg cctcggcctc tgagctattc
     8461 cagaagtagt gaggaggctt ttttggaggc ctttcaaata tgtatccgct catgagacaa
     8521 taaccctgat aaatgcttca ataatattga aaaaggaaga gtatgagtat tcaacatttc
     8581 cgtgtcgccc ttattccctt ttttgcggca ttttgccttc ctgtttttgc tcacccagaa
     8641 acgctggtga aagtaaaaga tgctgaagat cagttgggtg cacgagtggg ttacatcgaa
     8701 ctggatctca acagcggtaa gatccttgag agttttcgcc ccgaagaacg ttttccaatg
     8761 atgagcactt ttaaagttct gctatgtggc gcggtattat cccgtattga cgccgggcaa
     8821 gagcaactcg gtcgccgcat acactattct cagaatgact tggttgagta ctcaccagtc
     8881 acagaaaagc atcttacgga tggcatgaca gtaagagaat tatgcagtgc tgccataacc
     8941 atgagtgata acactgcggc caacttactt ctgacaacga tcggaggacc gaaggagcta
     9001 accgcttttt tgcacaacat gggggatcat gtaactcgcc ttgatcgttg ggaaccggag
     9061 ctgaatgaag ccataccaaa cgacgagcgt gacaccacga tgcctgtagc aatggcaaca
     9121 acgttgcgca aactattaac tggcgaacta cttactctag cttcccggca acaattaata
     9181 gactggatgg aggcggataa agttgcagga ccacttctgc gctcggccct tccggctggc
     9241 tggtttattg ctgataaatc tggagccggt gagcgtgggt ctcgcggtat cattgcagca
     9301 ctggggccag atggtaagcc ctcccgtatc gtagttatct acacgacggg gagtcaggca
     9361 actatggatg aacgaaatag acagatcgct gagataggtg cctcactgat taagcattgg
     9421 taactgtcag accaagttta ctcatatata ctttagattg atttaaaact tcatttttaa
     9481 tttaaaagga tctaggtgaa gatccttttt gataatctca tgaccaaaat cccttaacgt
     9541 gagttttcgt tccactgagc gtcagacccc gtagaaaaga tcaaaggatc ttcttgagat
     9601 cctttttttc tgcgcgtaat ctgctgcttg caaacaaaaa aaccaccgct accagcggtg
     9661 gtttgtttgc cggatcaaga gctaccaact ctttttccga aggtaactgg cttcagcaga
     9721 gcgcagatac caaatactgt tcttctagtg tagccgtagt taggccacca cttcaagaac
     9781 tctgtagcac cgcctacata cctcgctctg ctaatcctgt taccagtggc tgctgccagt
     9841 ggcgataagt cgtgtcttac cgggttggac tcaagacgat agttaccgga taaggcgcag
     9901 cggtcgggct gaacgggggg ttcgtgcaca cagcccagct tggagcgaac gacctacacc
     9961 gaactgagat acctacagcg tgagctatga gaaagcgcca cgcttcccga agggagaaag
    10021 gcggacaggt atccggtaag cggcagggtc ggaacaggag agcgcacgag ggagcttcca
    10081 gggggaaacg cctggtatct ttatagtcct gtcgggtttc gccacctctg acttgagcgt
    10141 cgatttttgt gatgctcgtc aggggggcgg agcctatgga aaaacgccag caacgcggcc
    10201 tttttacggt tcctggcctt ttgctggcct tttgctcaca tgttctttcc tgcgttatcc
    10261 cctgattctg tggataaccg tattaccgcc tttgagtgag ctgataccgc tcgccgcagc
    10321 cgaacgaccg agcgcagcga gtcagtgagc gaggaagcgg aagagcgccc aatacgcaaa
    10381 ccgcctctcc ccgcgcgttg gccgattcat taatgcagct ggcacgacag gtttcccgac
    10441 tggaaagcgg gcagtgagcg caacgcaatt aatgtgagtt agctcactca ttaggcaccc
    10501 caggctttac actttatgct tccggctcgt atgttgtgtg gaattgtgag cggataacaa
    10561 tttcacacag gaaacagcta tgaccatgat tacgccaagc gcgcaattaa ccctcactaa
    10621 agggaacaaa agctggagct gcaagcttaa tgtagtctta tgcaatactc ttgtagtctt
    10681 gcaacatggt aacgatgagt tagcaacatg ccttacaagg agagaaaaag caccgtgcat
    10741 gccgattggt ggaagtaagg tggtacgatc gtgccttatt aggaaggcaa cagacgggtc
    10801 tgacatggat tggacgaacc actgaattgc cgcattgcag agatattgta tttaagtgcc
    10861 tagctcgata cataaacggg tctctctggt tagaccagat ctgagcctgg gagctctctg
    10921 gctaactagg gaacccactg cttaagcctc aataaagctt gccttgagtg cttcaagtag
    10981 tgtgtgcccg tctgttgtgt gactctggta actagagatc cctcagaccc ttttagtcag
    11041 tgtggaaaat ctctagcagt ggcgcccgaa cagggacttg aaagcgaaag ggaaaccaga
    11101 ggagctctct cgacgcagga ctcggcttgc tgaagcgcgc acggcaagag gcgaggggcg
    11161 gcgactggtg agtacgccaa aaattttgac tagcggaggc tagaaggaga gagatgggtg
    11221 cgagagcgtc agtattaagc gggggagaat tagatcgcga tgggaaaaaa ttcggttaag
    11281 gccaggggga aagaaaaaat ataaattaaa acatatagta tgggcaagca gggagctaga
    11341 acgattcgca gttaatcctg gcctgttaga aacatcagaa ggctgtagac aaatactggg
    11401 acagctacaa ccatcccttc agacaggatc agaagaactt agatcattat ataatacagt
    11461 agcaaccctc tattgtgtgc atcaaaggat agagataaaa gacaccaagg aagctttaga
    11521 caagatagag gaagagcaaa acaaaagtaa gaccaccgca cagcaagcgg ccgctgatct
    11581 tcagacctgg aggaggagat atgagggaca attggagaag tgaattatat aaatataaag
    11641 tagtaaaaat tgaaccatta ggagtagcac ccaccaaggc aaagagaaga gtggtgcaga
    11701 gagaaaaaag agcagtggga ataggagctt tgttccttgg gttcttggga gcagcaggaa
    11761 gcactatggg cgcagcgtca atgacgctga cggtacaggc cagacaatta ttgtctggta
    11821 tagtgcagca gcagaacaat ttgctgaggg ctattgaggc gcaacagcat ctgttgcaac
    11881 tcacagtctg gggcatcaag cagctccagg caagaatcct ggctgtggaa agatacctaa
    11941 aggatcaaca gctcctgggg atttggggtt gctctggaaa actcatttgc accactgctg
    12001 tgccttggaa tgctagttgg agtaataaat ctctggaaca gatttggaat cacacgacct
    12061 ggatggagtg ggacagagaa attaacaatt acacaagctt aatacactcc ttaattgaag
    12121 aatcgcaaaa ccagcaagaa aagaatgaac aagaattatt ggaattagat aaatgggcaa
    12181 gtttgtggaa ttggtttaac ataacaaatt ggctgtggta tataaaatta ttcataatga
    12241 tagtaggagg cttggtaggt ttaagaatag tttttgctgt actttctata gtgaatagag
    12301 ttaggcaggg atattcacca ttatcgtttc agacccacct cccaaccccg aggggacaat
    12361 tctcgacctc gagacaaatg gcagtattca tccacaattt taaaagaaaa ggggggattg
    12421 gggggtacag tgcaggggaa agaatagtag acataatagc aacagacata caaactaaag
    12481 aattacaaaa acaaattaca aaaattcaaa attttcgggt ttattacagg gacagcagag
    12541 atccactttg gccgcgaatc gatatgtcga gtttac
//
