LOCUS       sequence-325304-       13773 bp    DNA     circular SYN 15-DEC-2025
DEFINITION  3rd generation lentivirus transfer vector for Cre-removable Cas9, 
            GFP reporter, and hygro resistance.
ACCESSION   .
VERSION     .
KEYWORDS    .
SOURCE      synthetic DNA construct
  ORGANISM  synthetic DNA construct
REFERENCE   1  (bases 1 to 13773)
  AUTHORS   Dubrot J, Lane-Reticker SK, Kessler EA, Ayer A, Mishra G, Wolfe CH, 
            Zimmer MD, Du PP, Mahapatra A, Ockerman KM, Davis TGR, Kohnle IC, 
            Pope HW, Allen PM, Olander KE, Iracheta-Vellve A, Doench JG, Haining
            WN, Yates KB, Manguso RT
  TITLE     In vivo screens using a selective CRISPR antigen removal lentiviral 
            vector system reveal immune dependencies in renal cell carcinoma.
  JOURNAL   Immunity. 2021 Jan 20. pii: S1074-7613(21)00001-7. doi: 
            10.1016/j.immuni.2021.01.001.
  PUBMED    33497609
REFERENCE   2  (bases 1 to 13773)
  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: "Immunity. 
            2021 Jan 20. pii: S1074-7613(21)00001-7. doi: 
            10.1016/j.immuni.2021.01.001."
COMMENT     Sequence Label: pSCAR_Cas9-hygro_GFP
FEATURES             Location/Qualifiers
     source          1..13773
                     /mol_type="other DNA"
                     /organism="synthetic DNA construct"
     CDS             16..36
                     /codon_start=1
                     /product="nuclear localization signal of SV40 (simian virus
                     40) large T antigen"
                     /label=SV40 NLS
                     /translation="PKKKRKV"
     CDS             43..4143
                     /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             43..4143
                     /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             4150..4197
                     /codon_start=1
                     /product="bipartite nuclear localization signal from 
                     nucleoplasmin"
                     /label=nucleoplasmin NLS
                     /translation="KRPAATKKAGQAKKKK"
     CDS             4219..4275
                     /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             4282..5298
                     /codon_start=1
                     /product="hygromycin B phosphotransferase"
                     /label=HygR
                     /note="confers resistance to hygromycin"
                     /translation="KKPELTATSVEKFLIEKFDSVSDLMQLSEGEESRAFSFDVGGRGY
                     VLRVNSCADGFYKDRYVYRHFASAALPIPEVLDIGEFSESLTYCISRRAQGVTLQDLPE
                     TELPAVLQPVAEAMDAIAAADLSQTSGFGPFGPQGIGQYTTWRDFICAIADPHVYHWQT
                     VMDDTVSASVAQALDELMLWAEDCPEVRHLVHADFGSNNVLTDNGRITAVIDWSEAMFG
                     DSQYEVANIFFWRPWLACMEQQTRYFERRHPELAGSPRLRAYMLRIGLDQLYQSLVDGN
                     FDDAAWAQGRCDAIVRSGAGTVGRTQIARRSAAVWTDGCVEVLADSGNRRPSTRPRAK"
     misc_feature    5336..5924
                     /label=WPRE
                     /note="woodchuck hepatitis virus posttranscriptional 
                     regulatory element"
     primer_bind     complement(5389..5409)
                     /label=WPRE-R
                     /note="WPRE, reverse primer"
     CDS             complement(5807..5818)
                     /codon_start=1
                     /product="Factor Xa recognition and cleavage site"
                     /label=Factor Xa site
                     /translation="IEGR"
     protein_bind    complement(6051..6084)
                     /label=loxP
                     /bound_moiety="Cre recombinase"
                     /note="Cre-mediated recombination occurs in the 8-bp core 
                     sequence (ATGTATGC) (Shaw et al., 2021)."
     LTR             6104..6284
                     /label=5' LTR (truncated)
                     /note="truncated 5' long terminal repeat (LTR) from HIV-1"
     polyA_signal    6361..6482
                     /label=SV40 poly(A) signal
                     /note="SV40 polyadenylation signal"
     primer_bind     complement(6398..6417)
                     /label=SV40pA-R
                     /note="SV40 polyA, reverse primer"
     primer_bind     6452..6471
                     /label=EBV-rev
                     /note="SV40 polyA terminator, reverse primer"
     rep_origin      6522..6657
                     /label=SV40 ori
                     /note="SV40 origin of replication"
     primer_bind     complement(6677..6696)
                     /label=T7
                     /note="T7 promoter, forward primer"
     promoter        complement(6678..6696)
                     /label=T7 promoter
                     /note="promoter for bacteriophage T7 RNA polymerase"
     primer_bind     complement(6706..6723)
                     /label=M13 Forward
                     /note="In lacZ gene. Also called M13-F20 or M13 (-21) 
                     Forward"
     primer_bind     complement(6706..6722)
                     /label=M13 fwd
                     /note="common sequencing primer, one of multiple similar 
                     variants"
     primer_bind     complement(6715..6737)
                     /label=M13/pUC Forward
                     /note="In lacZ gene"
     rep_origin      6864..7319
                     /direction=RIGHT
                     /label=f1 ori
                     /note="f1 bacteriophage origin of replication; arrow 
                     indicates direction of (+) strand synthesis"
     primer_bind     complement(6951..6970)
                     /label=F1ori-R
                     /note="F1 origin, reverse primer"
     primer_bind     7161..7182
                     /label=F1ori-F
                     /note="F1 origin, forward primer"
     promoter        7345..7449
                     /gene="bla"
                     /label=AmpR promoter
     CDS             join(7450..7518,7519..8310)
                     /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(7668..7687)
                     /label=Amp-R
                     /note="Ampicillin resistance gene, reverse primer"
     rep_origin      8481..9069
                     /direction=RIGHT
                     /label=ori
                     /note="high-copy-number ColE1/pMB1/pBR322/pUC origin of 
                     replication"
     primer_bind     8970..8989
                     /label=pBR322ori-F
                     /note="pBR322 origin, forward primer"
     primer_bind     9223..9240
                     /label=L4440
                     /note="L4440 vector, forward primer"
     protein_bind    9357..9378
                     /label=CAP binding site
                     /bound_moiety="E. coli catabolite activator protein"
                     /note="CAP binding activates transcription in the presence 
                     of cAMP."
     promoter        join(9393..9398,9399..9416,9417..9423)
                     /label=lac promoter
                     /note="promoter for the E. coli lac operon"
     protein_bind    9431..9447
                     /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     9436..9458
                     /label=M13/pUC Reverse
                     /note="In lacZ gene"
     primer_bind     9455..9471
                     /label=M13 rev
                     /note="common sequencing primer, one of multiple similar 
                     variants"
     primer_bind     9455..9471
                     /label=M13 Reverse
                     /note="In lacZ gene. Also called M13-rev"
     primer_bind     9490..9510
                     /label=T3
                     /note="T3 promoter, forward primer"
     promoter        9492..9510
                     /label=T3 promoter
                     /note="promoter for bacteriophage T3 RNA polymerase"
     promoter        9538..9764
                     /label=RSV promoter
                     /note="Rous sarcoma virus enhancer/promoter"
     LTR             9765..9945
                     /label=5' LTR (truncated)
                     /note="truncated 5' long terminal repeat (LTR) from HIV-1"
     misc_feature    9992..10117
                     /label=HIV-1 Psi
                     /note="packaging signal of human immunodeficiency virus 
                     type 1"
     misc_feature    10610..10843
                     /label=RRE
                     /note="The Rev response element (RRE) of HIV-1 allows for 
                     Rev-dependent mRNA export from the nucleus to the 
                     cytoplasm."
     CDS             11028..11072
                     /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"
     promoter        11303..11513
                     /label=SV40 promoter
                     /note="SV40 early promoter"
     promoter        11325..11521
                     /label=SV40 promoter
                     /note="SV40 early promoter"
     rep_origin      11372..11507
                     /label=SV40 ori
                     /note="SV40 origin of replication"
     CDS             join(11543..11545,11546..11548,11549..12265)
                     /codon_start=1
                     /product="the original enhanced GFP (Yang et al., 1996)"
                     /label=EGFP
                     /note="mammalian codon-optimized"
                     /translation="MVSKGEELFTGVVPILVELDGDVNGHKFSVSGEGEGDATYGKLTL
                     KFICTTGKLPVPWPTLVTTLTYGVQCFSRYPDHMKQHDFFKSAMPEGYVQERTIFFKDD
                     GNYKTRAEVKFEGDTLVNRIELKGIDFKEDGNILGHKLEYNYNSHNVYIMADKQKNGIK
                     VNFKIRHNIEDGSVQLADHYQQNTPIGDGPVLLPDNHYLSTQSALSKDPNEKRDHMVLL
                     EFVTAAGITLGMDELYKS"
     primer_bind     complement(11849..11868)
                     /label=EXFP-R
                     /note="For distinguishing EGFP variants, reverse primer"
     primer_bind     12196..12217
                     /label=EGFP-C
                     /note="EGFP, forward primer"
     misc_feature    12321..12438
                     /label=cPPT/CTS
                     /note="central polypurine tract and central termination 
                     sequence of HIV-1"
     promoter        12576..13754
                     /label=EF-1-alpha promoter
                     /note="strong constitutive promoter for human elongation 
                     factor EF-1-alpha"
     intron          12807..13745
                     /label=EF-1-alpha intron A
                     /note="intron upstream of the start codon of human 
                     EF-1-alpha"
     primer_bind     13702..13722
                     /label=EF1a-F
                     /note="Human elongation factor-1a promoter, forward primer"
ORIGIN
        1 ggcttcacca tggcaccaaa gaagaagcgg aaggtcgcag ccgacaagaa gtacagcatc
       61 ggcctggaca tcggcaccaa ctctgtgggc tgggccgtga tcaccgacga gtacaaggtg
      121 cccagcaaga aattcaaggt gctgggcaac accgaccggc acagcatcaa gaagaacctg
      181 atcggagccc tgctgttcga cagcggcgaa acagccgagg ccacccggct gaagagaacc
      241 gccagaagaa gatacaccag acggaagaac cggatctgct atctgcaaga gatcttcagc
      301 aacgagatgg ccaaggtgga cgacagcttc ttccacagac tggaagagtc cttcctggtg
      361 gaagaggata agaagcacga gcggcacccc atcttcggca acatcgtgga cgaggtggcc
      421 taccacgaga agtaccccac catctaccac ctgagaaaga aactggtgga cagcaccgac
      481 aaggccgacc tgcggctgat ctatctggcc ctggcccaca tgatcaagtt ccggggccac
      541 ttcctgatcg agggcgacct gaaccccgac aacagcgacg tggacaagct gttcatccag
      601 ctggtgcaga cctacaacca gctgttcgag gaaaacccca tcaacgccag cggcgtagac
      661 gccaaggcca tcctgtctgc cagactgagc aagagcagac ggctggaaaa tctgatcgcc
      721 cagctgcccg gcgagaagaa gaatggcctg ttcggaaacc tgattgccct gagcctgggc
      781 ctgaccccca acttcaagag caacttcgac ctggccgagg atgccaaact gcagctgagc
      841 aaggacacct acgacgacga cctggacaac ctgctggccc agatcggcga ccagtacgcc
      901 gacctgtttc tggccgccaa gaacctgtcc gacgccatcc tgctgagcga catcctgaga
      961 gtgaacaccg agatcaccaa ggcccccctg agcgcctcta tgatcaagag atacgacgag
     1021 caccaccagg acctgaccct gctgaaagct ctcgtgcggc agcagctgcc tgagaagtac
     1081 aaagagattt tcttcgacca gagcaagaac ggctacgccg gctacattga cggcggagcc
     1141 agccaggaag agttctacaa gttcatcaag cccatcctgg aaaagatgga cggcaccgag
     1201 gaactgctcg tgaagctgaa cagagaggac ctgctgcgga agcagcgtac gttcgacaac
     1261 ggcagcatcc cccaccagat ccacctggga gagctgcacg ccattctgcg gcggcaggaa
     1321 gatttttacc cattcctgaa ggacaaccgg gaaaagatcg agaagatcct gaccttccgc
     1381 atcccctact acgtgggccc tctggccagg ggaaacagca gattcgcctg gatgaccaga
     1441 aagagcgagg aaaccatcac cccctggaac ttcgaggaag tggtggacaa gggcgccagc
     1501 gcccagagct tcatcgagcg gatgaccaac ttcgataaga acctgcccaa cgagaaggtg
     1561 ctgcccaagc acagcctgct gtacgagtac ttcaccgtgt ataacgagct gaccaaagtg
     1621 aaatacgtga ccgagggaat gagaaagccc gccttcctga gcggcgagca gaaaaaggcc
     1681 atcgtggacc tgctgttcaa gaccaaccgg aaagtgaccg tgaagcagct gaaagaggac
     1741 tacttcaaga aaatcgagtg cttcgactcc gtggaaatct ccggcgtgga agatcggttc
     1801 aacgcctccc tgggcacata ccacgatctg ctgaaaatta tcaaggacaa ggacttcctg
     1861 gacaatgagg aaaacgagga cattctggaa gatatcgtgc tgaccctgac actgtttgag
     1921 gacagagaga tgatcgagga acggctgaaa acctatgccc acctgttcga cgacaaagtg
     1981 atgaagcagc tgaagcggcg gagatacacc ggctggggca ggctgagccg gaagctgatc
     2041 aacggcatcc gggacaagca gtccggcaag acaatcctgg atttcctgaa gtccgacggc
     2101 ttcgccaaca gaaacttcat gcagctgatc cacgacgaca gcctgacctt taaagaggac
     2161 atccagaaag cccaggtgtc cggccagggc gatagcctgc acgagcacat tgccaatctg
     2221 gccggcagcc ccgccattaa gaagggcatc ctgcagacag tgaaggtggt ggacgagctc
     2281 gtgaaagtga tgggccggca caagcccgag aacatcgtga tcgaaatggc cagagagaac
     2341 cagaccaccc agaagggaca gaagaacagc cgcgagagaa tgaagcggat cgaagagggc
     2401 atcaaagagc tgggcagcca gatcctgaaa gaacaccccg tggaaaacac ccagctgcag
     2461 aacgagaagc tgtacctgta ctacctgcag aatgggcggg atatgtacgt ggaccaggaa
     2521 ctggacatca accggctgtc cgactacgat gtggaccata tcgtgcctca gagctttctg
     2581 aaggacgact ccatcgacaa caaggtgctg accagaagcg acaagaaccg gggcaagagc
     2641 gacaacgtgc cctccgaaga ggtcgtgaag aagatgaaga actactggcg gcagctgctg
     2701 aacgccaagc tgattaccca gagaaagttc gacaatctga ccaaggccga gagaggcggc
     2761 ctgagcgaac tggataaggc cggcttcatc aagagacagc tggtggaaac ccggcagatc
     2821 acaaagcacg tggcacagat cctggactcc cggatgaaca ctaagtacga cgagaatgac
     2881 aagctgatcc gggaagtgaa agtgatcacc ctgaagtcca agctggtgtc cgatttccgg
     2941 aaggatttcc agttttacaa agtgcgcgag atcaacaact accaccacgc ccacgacgcc
     3001 tacctgaacg ccgtcgtggg aaccgccctg atcaaaaagt accctaagct ggaaagcgag
     3061 ttcgtgtacg gcgactacaa ggtgtacgac gtgcggaaga tgatcgccaa gagcgagcag
     3121 gaaatcggca aggctaccgc caagtacttc ttctacagca acatcatgaa ctttttcaag
     3181 accgagatta ccctggccaa cggcgagatc cggaagcggc ctctgatcga gacaaacggc
     3241 gaaaccgggg agatcgtgtg ggataagggc cgggattttg ccaccgtgcg gaaagtgctg
     3301 agcatgcccc aagtgaatat cgtgaaaaag accgaggtgc agacaggcgg cttcagcaaa
     3361 gagtctatcc tgcccaagag gaacagcgat aagctgatcg ccagaaagaa ggactgggac
     3421 cctaagaagt acggcggctt cgacagcccc accgtggcct attctgtgct ggtggtggcc
     3481 aaagtggaaa agggcaagtc caagaaactg aagagtgtga aagagctgct ggggatcacc
     3541 atcatggaaa gaagcagctt cgagaagaat cccatcgact ttctggaagc caagggctac
     3601 aaagaagtga aaaaggacct gatcatcaag ctgcctaagt actccctgtt cgagctggaa
     3661 aacggccgga agagaatgct ggcctctgcc ggcgaactgc agaagggaaa cgaactggcc
     3721 ctgccctcca aatatgtgaa cttcctgtac ctggccagcc actatgagaa gctgaagggc
     3781 tcccccgagg ataatgagca gaaacagctg tttgtggaac agcacaagca ctacctggac
     3841 gagatcatcg agcagatcag cgagttctcc aagagagtga tcctggccga cgctaatctg
     3901 gacaaagtgc tgtccgccta caacaagcac cgggataagc ccatcagaga gcaggccgag
     3961 aatatcatcc acctgtttac cctgaccaat ctgggagccc ctgccgcctt caagtacttt
     4021 gacaccacca tcgaccggaa gaggtacacc agcaccaaag aggtgctgga cgccaccctg
     4081 atccaccaga gcatcaccgg cctgtacgag acacgaatcg atttgtctca gctgggaggc
     4141 gacgccgcaa aaaggccggc ggccacgaaa aaggccggcc aggcaaaaaa gaaaaaggca
     4201 ggctcaggcg gatccggcgc aacaaacttc tctctgctga aacaagccgg agatgtcgaa
     4261 gagaatcctg gaccgagcgc taaaaagcct gaactcaccg ctacctctgt cgagaagttt
     4321 ctgatcgaaa agttcgacag cgtctccgac ctgatgcagc tctccgaggg cgaagaatct
     4381 cgggctttca gcttcgatgt gggagggcgt ggatatgtcc tgcgggtgaa tagctgcgcc
     4441 gatggtttct acaaagatcg ctatgtttat cggcactttg catccgccgc tctccctatt
     4501 cccgaagtgc ttgacattgg ggagttcagc gagagcctga cctattgcat ctcccgccgt
     4561 gcacagggtg tcaccttgca agacctgcct gaaaccgaac tgcccgctgt tctccagccc
     4621 gtcgccgagg ccatggatgc catcgctgcc gccgatctta gccagaccag cgggttcggc
     4681 ccattcggac ctcaaggaat cggtcaatac actacatggc gcgatttcat ctgcgctatt
     4741 gctgatcccc atgtgtatca ctggcaaact gtgatggacg acaccgtcag tgcctccgtc
     4801 gcccaggctc tcgatgagct gatgctttgg gccgaggact gccccgaagt ccggcacctc
     4861 gtgcacgccg atttcggctc caacaatgtc ctgaccgaca atggccgcat aacagccgtc
     4921 attgactgga gcgaggccat gttcggggat tcccaatacg aggtcgccaa catcttcttc
     4981 tggaggccct ggttggcttg tatggagcag cagacccgct acttcgagcg gaggcatccc
     5041 gagcttgcag gatctcctcg gctccgggct tatatgctcc gcattggtct tgaccaactc
     5101 tatcagagct tggttgacgg caatttcgat gatgcagctt gggctcaggg tcgctgcgac
     5161 gcaatcgtcc ggtccggagc cgggactgtc gggcgtacac aaatcgcccg cagaagcgct
     5221 gccgtctgga ccgatggctg tgtggaagtg ctcgccgata gtggaaacag acgccccagc
     5281 actcgtccta gggcaaagga tctgcagtga aaggacatca acgcgttaag tcracaatca
     5341 acctctggat tacaaaattt gtgaaagatt gactggtatt cttaactatg ttgctccttt
     5401 tacgctatgt ggatacgctg ctttaatgcc tttgtatcat gctattgctt cccgtatggc
     5461 tttcattttc tcctccttgt ataaatcctg gttgctgtct ctttatgagg agttgtggcc
     5521 cgttgtcagg caacgtggcg tggtgtgcac tgtgtttgct gacgcaaccc ccactggttg
     5581 gggcattgcc accacctgtc agctcctttc cgggactttc gctttccccc tccctattgc
     5641 cacggcggaa ctcatcgccg cctgccttgc ccgctgctgg acaggggctc ggctgttggg
     5701 cactgacaat tccgtggtgt tgtcggggaa atcatcgtcc tttccttggc tgctcgcctg
     5761 tgttgccacc tggattctgc gcgggacgtc cttctgctac gtcccttcgg ccctcaatcc
     5821 agcggacctt ccttcccgcg gcctgctgcc ggctctgcgg cctcttccgc gtcttcgcct
     5881 tcgccctcag acgagtcgga tctccctttg ggccgcctcc ccgcgtcgac tttaagacca
     5941 atgacttaca aggcagctgt agatcttagc cactttttaa aagaaaaggg gggactggaa
     6001 gggctaattc actcccaacg aagacaagat gggatcaatt caccatggga ataacttcgt
     6061 atagcataca ttatacgaag ttatgctgct ttttgcttgt actgggtctc tctggttaga
     6121 ccagatctga gcctgggagc tctctggcta actagggaac ccactgctta agcctcaata
     6181 aagcttgcct tgagtgcttc aagtagtgtg tgcccgtctg ttgtgtgact ctggtaacta
     6241 gagatccctc agaccctttt agtcagtgtg gaaaatctct agcatacgta tagtagttca
     6301 tgtcatctta ttattcagta tttataactt gcaaagaaat gaatatcaga gagtgagagg
     6361 aacttgttta ttgcagctta taatggttac aaataaagca atagcatcac aaatttcaca
     6421 aataaagcat ttttttcact gcattctagt tgtggtttgt ccaaactcat caatgtatct
     6481 tatcatgtct ggctctagct atcccgcccc taactccgcc catcccgccc ctaactccgc
     6541 ccagttccgc ccattctccg ccccatggct gactaatttt ttttatttat gcagaggccg
     6601 aggccgcctc ggcctctgag ctattccaga agtagtgagg aggctttttt ggaggcctag
     6661 ggacgtaccc aattcgccct atagtgagtc gtattacgcg cgctcactgg ccgtcgtttt
     6721 acaacgtcgt gactgggaaa accctggcgt tacccaactt aatcgccttg cagcacatcc
     6781 ccctttcgcc agctggcgta atagcgaaga ggcccgcacc gatcgccctt cccaacagtt
     6841 gcgcagcctg aatggcgaat gggacgcgcc ctgtagcggc gcattaagcg cggcgggtgt
     6901 ggtggttacg cgcagcgtga ccgctacact tgccagcgcc ctagcgcccg ctcctttcgc
     6961 tttcttccct tcctttctcg ccacgttcgc cggctttccc cgtcaagctc taaatcgggg
     7021 gctcccttta gggttccgat ttagtgcttt acggcacctc gaccccaaaa aacttgatta
     7081 gggtgatggt tcacgtagtg ggccatcgcc ctgatagacg gtttttcgcc ctttgacgtt
     7141 ggagtccacg ttctttaata gtggactctt gttccaaact ggaacaacac tcaaccctat
     7201 ctcggtctat tcttttgatt tataagggat tttgccgatt tcggcctatt ggttaaaaaa
     7261 tgagctgatt taacaaaaat ttaacgcgaa ttttaacaaa atattaacgc ttacaattta
     7321 ggtggcactt ttcggggaaa tgtgcgcgga acccctattt gtttattttt ctaaatacat
     7381 tcaaatatgt atccgctcat gagacaataa ccctgataaa tgcttcaata atattgaaaa
     7441 aggaagagta tgagtattca acatttccgt gtcgccctta ttcccttttt tgcggcattt
     7501 tgccttcctg tttttgctca cccagaaacg ctggtgaaag taaaagatgc tgaagatcag
     7561 ttgggtgcac gagtgggtta catcgaactg gatctcaaca gcggtaagat ccttgagagt
     7621 tttcgccccg aagaacgttt tccaatgatg agcactttta aagttctgct atgtggcgcg
     7681 gtattatccc gtattgacgc cgggcaagag caactcggtc gccgcataca ctattctcag
     7741 aatgacttgg ttgagtactc accagtcaca gaaaagcatc ttacggatgg catgacagta
     7801 agagaattat gcagtgctgc cataaccatg agtgataaca ctgcggccaa cttacttctg
     7861 acaacgatcg gaggaccgaa ggagctaacc gcttttttgc acaacatggg ggatcatgta
     7921 actcgccttg atcgttggga accggagctg aatgaagcca taccaaacga cgagcgtgac
     7981 accacgatgc ctgtagcaat ggcaacaacg ttgcgcaaac tattaactgg cgaactactt
     8041 actctagctt cccggcaaca attaatagac tggatggagg cggataaagt tgcaggacca
     8101 cttctgcgct cggcccttcc ggctggctgg tttattgctg ataaatctgg agccggtgag
     8161 cgtgggtctc gcggtatcat tgcagcactg gggccagatg gtaagccctc ccgtatcgta
     8221 gttatctaca cgacggggag tcaggcaact atggatgaac gaaatagaca gatcgctgag
     8281 ataggtgcct cactgattaa gcattggtaa ctgtcagacc aagtttactc atatatactt
     8341 tagattgatt taaaacttca tttttaattt aaaaggatct aggtgaagat cctttttgat
     8401 aatctcatga ccaaaatccc ttaacgtgag ttttcgttcc actgagcgtc agaccccgta
     8461 gaaaagatca aaggatcttc ttgagatcct ttttttctgc gcgtaatctg ctgcttgcaa
     8521 acaaaaaaac caccgctacc agcggtggtt tgtttgccgg atcaagagct accaactctt
     8581 tttccgaagg taactggctt cagcagagcg cagataccaa atactgttct tctagtgtag
     8641 ccgtagttag gccaccactt caagaactct gtagcaccgc ctacatacct cgctctgcta
     8701 atcctgttac cagtggctgc tgccagtggc gataagtcgt gtcttaccgg gttggactca
     8761 agacgatagt taccggataa ggcgcagcgg tcgggctgaa cggggggttc gtgcacacag
     8821 cccagcttgg agcgaacgac ctacaccgaa ctgagatacc tacagcgtga gctatgagaa
     8881 agcgccacgc ttcccgaagg gagaaaggcg gacaggtatc cggtaagcgg cagggtcgga
     8941 acaggagagc gcacgaggga gcttccaggg ggaaacgcct ggtatcttta tagtcctgtc
     9001 gggtttcgcc acctctgact tgagcgtcga tttttgtgat gctcgtcagg ggggcggagc
     9061 ctatggaaaa acgccagcaa cgcggccttt ttacggttcc tggccttttg ctggcctttt
     9121 gctcacatgt tctttcctgc gttatcccct gattctgtgg ataaccgtat taccgccttt
     9181 gagtgagctg ataccgctcg ccgcagccga acgaccgagc gcagcgagtc agtgagcgag
     9241 gaagcggaag agcgcccaat acgcaaaccg cctctccccg cgcgttggcc gattcattaa
     9301 tgcagctggc acgacaggtt tcccgactgg aaagcgggca gtgagcgcaa cgcaattaat
     9361 gtgagttagc tcactcatta ggcaccccag gctttacact ttatgcttcc ggctcgtatg
     9421 ttgtgtggaa ttgtgagcgg ataacaattt cacacaggaa acagctatga ccatgattac
     9481 gccaagcgcg caattaaccc tcactaaagg gaacaaaagc tggagctgca agcttaatgt
     9541 agtcttatgc aatactcttg tagtcttgca acatggtaac gatgagttag caacatgcct
     9601 tacaaggaga gaaaaagcac cgtgcatgcc gattggtgga agtaaggtgg tacgatcgtg
     9661 ccttattagg aaggcaacag acgggtctga catggattgg acgaaccact gaattgccgc
     9721 attgcagaga tattgtattt aagtgcctag ctcgatacat aaacgggtct ctctggttag
     9781 accagatctg agcctgggag ctctctggct aactagggaa cccactgctt aagcctcaat
     9841 aaagcttgcc ttgagtgctt caagtagtgt gtgcccgtct gttgtgtgac tctggtaact
     9901 agagatccct cagacccttt tagtcagtgt ggaaaatctc tagcagtggc gcccgaacag
     9961 ggacttgaaa gcgaaaggga aaccagagga gctctctcga cgcaggactc ggcttgctga
    10021 agcgcgcacg gcaagaggcg aggggcggcg actggtgagt acgccaaaaa ttttgactag
    10081 cggaggctag aaggagagag atgggtgcga gagcgtcagt attaagcggg ggagaattag
    10141 atcgcgatgg gaaaaaattc ggttaaggcc agggggaaag aaaaaatata aattaaaaca
    10201 tatagtatgg gcaagcaggg agctagaacg attcgcagtt aatcctggcc tgttagaaac
    10261 atcagaaggc tgtagacaaa tactgggaca gctacaacca tcccttcaga caggatcaga
    10321 agaacttaga tcattatata atacagtagc aaccctctat tgtgtgcatc aaaggataga
    10381 gataaaagac accaaggaag ctttagacaa gatagaggaa gagcaaaaca aaagtaagac
    10441 caccgcacag caagcggccg ctgatcttca gacctggagg aggagatatg agggacaatt
    10501 ggagaagtga attatataaa tataaagtag taaaaattga accattagga gtagcaccca
    10561 ccaaggcaaa gagaagagtg gtgcagagag aaaaaagagc agtgggaata ggagctttgt
    10621 tccttgggtt cttgggagca gcaggaagca ctatgggcgc agcgtcaatg acgctgacgg
    10681 tacaggccag acaattattg tctggtatag tgcagcagca gaacaatttg ctgagggcta
    10741 ttgaggcgca acagcatctg ttgcaactca cagtctgggg catcaagcag ctccaggcaa
    10801 gaatcctggc tgtggaaaga tacctaaagg atcaacagct cctggggatt tggggttgct
    10861 ctggaaaact catttgcacc actgctgtgc cttggaatgc tagttggagt aataaatctc
    10921 tggaacagat ttggaatcac acgacctgga tggagtggga cagagaaatt aacaattaca
    10981 caagcttaat acactcctta attgaagaat cgcaaaacca gcaagaaaag aatgaacaag
    11041 aattattgga attagataaa tgggcaagtt tgtggaattg gtttaacata acaaattggc
    11101 tgtggtatat aaaattattc ataatgatag taggaggctt ggtaggttta agaatagttt
    11161 ttgctgtact ttctatagtg aatagagtta ggcagggata ttcaccatta tcgtttcaga
    11221 cccacctccc aaccccgagg ggacccatgc attgcatctc aattagtcag caaccaggtg
    11281 tggaaagtcc ccaggctccc cagcaggcag aagtatgcaa agcatgcgtc tcaattagtc
    11341 agcaaccata gtcccgcccc taactccgcc catcccgccc ctaactccgc ccagttccgc
    11401 ccattctccg ccccatggct gactaatttt ttttatttat gcagaggccg aggccgcctc
    11461 ggcctctgag ctattccaga agtagtgagg aggctttttt ggaggcctag gcttttgcaa
    11521 aaagctttct agaggtacca ccatggtgag caagggcgag gagctgttca ccggggtggt
    11581 gcccatcctg gtcgagctgg acggcgacgt aaacggccac aagttcagcg tgtctggcga
    11641 gggcgagggc gatgccacct acggcaagct gaccctgaag ttcatctgca ccaccggcaa
    11701 gctgcccgtg ccctggccca ccctcgtgac caccctgacc tacggcgtgc agtgcttcag
    11761 ccgctacccc gaccacatga agcagcacga cttcttcaag tccgccatgc ccgaaggcta
    11821 cgtccaggag cgcaccatct tcttcaagga cgacggcaac tacaagaccc gcgccgaggt
    11881 gaagttcgag ggcgacaccc tggtgaaccg catcgagctg aagggcatcg acttcaagga
    11941 ggacggcaac atcctggggc acaagctgga gtacaactac aacagccaca acgtctatat
    12001 catggccgac aagcagaaga acggcatcaa ggtgaacttc aagatccgcc acaacatcga
    12061 ggacggcagc gtgcagctcg ccgaccacta ccagcagaac acccccatcg gcgacggccc
    12121 cgtgctgctg cccgacaacc actacctgag cacccagtcc gccctgagca aagaccccaa
    12181 cgagaagcgc gatcacatgg tcctgctgga gttcgtgacc gccgccggga tcactctcgg
    12241 catggacgag ctgtacaagt cctaaggcgc gccgttaacg aattctagat cttgagacaa
    12301 atggcagtat tcatccacaa ttttaaaaga aaagggggga ttggggggta cagtgcaggg
    12361 gaaagaatag tagacataat agcaacagac atacaaacta aagaattaca aaaacaaatt
    12421 acaaaaattc aaaattttcg ggtttattac agggacagca gagatccact ttggcgccgg
    12481 ctcgaggcct gcaggtgcaa agatggataa agttttaaac agagaggaat ctttgcagct
    12541 aatggacctt ctaggtcttg aaaggagtgg gaattggctc cggtgcccgt cagtgggcag
    12601 agcgcacatc gcccacagtc cccgagaagt tggggggagg ggtcggcaat tgaaccggtg
    12661 cctagagaag gtggcgcggg gtaaactggg aaagtgatgt cgtgtactgg ctccgccttt
    12721 ttcccgaggg tgggggagaa ccgtatataa gtgcagtagt cgccgtgaac gttctttttc
    12781 gcaacgggtt tgccgccaga acacaggtaa gtgccgtgtg tggttcccgc gggcctggcc
    12841 tctttacggg ttatggccct tgcgtgcctt gaattacttc cacctggctg cagtacgtga
    12901 ttcttgatcc cgagcttcgg gttggaagtg ggtgggagag ttcgaggcct tgcgcttaag
    12961 gagccccttc gcctcgtgct tgagttgagg cctggcctgg gcgctggggc cgccgcgtgc
    13021 gaatctggtg gcaccttcgc gcctgtctcg ctgctttcga taagtctcta gccatttaaa
    13081 atttttgatg acctgctgcg acgctttttt tctggcaaga tagtcttgta aatgcgggcc
    13141 aagatctgca cactggtatt tcggtttttg gggccgcggg cggcgacggg gcccgtgcgt
    13201 cccagcgcac atgttcggcg aggcggggcc tgcgagcgcg gccaccgaga atcggacggg
    13261 ggtagtctca agctggccgg cctgctctgg tgcctggcct cgcgccgccg tgtatcgccc
    13321 cgccctgggc ggcaaggctg gcccggtcgg caccagttgc gtgagcggaa agatggccgc
    13381 ttcccggccc tgctgcaggg agctcaaaat ggaggacgcg gcgctcggga gagcgggcgg
    13441 gtgagtcacc cacacaaagg aaaagggcct ttccgtcctc agccgtcgct tcatgtgact
    13501 ccacggagta ccgggcgccg tccaggcacc tcgattagtt ctcgagcttt tggagtacgt
    13561 cgtctttagg ttggggggag gggttttatg cgatggagtt tccccacact gagtgggtgg
    13621 agactgaagt taggccagct tggcacttga tgtaattctc cttggaattt gccctttttg
    13681 agtttggatc ttggttcatt ctcaagcctc agacagtggt tcaaagtttt tttcttccat
    13741 ttcaggtgtc gtgaggctag catcgattga tca
//
