LOCUS       sequence-471574-       13264 bp    DNA     circular SYN 15-DEC-2025
DEFINITION  3rd generation lentivirus transfer vector for Cre-removable Cas9, 
            GFP reporter, and blast resistance.
ACCESSION   .
VERSION     .
KEYWORDS    .
SOURCE      synthetic DNA construct
  ORGANISM  synthetic DNA construct
REFERENCE   1  (bases 1 to 13264)
  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 13264)
  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-blast_GFP
FEATURES             Location/Qualifiers
     source          1..13264
                     /mol_type="other DNA"
                     /organism="synthetic DNA construct"
     rep_origin      659..1247
                     /direction=RIGHT
                     /label=ori
                     /note="high-copy-number ColE1/pMB1/pBR322/pUC origin of 
                     replication"
     primer_bind     1148..1167
                     /label=pBR322ori-F
                     /note="pBR322 origin, forward primer"
     primer_bind     1401..1418
                     /label=L4440
                     /note="L4440 vector, forward primer"
     protein_bind    1535..1556
                     /label=CAP binding site
                     /bound_moiety="E. coli catabolite activator protein"
                     /note="CAP binding activates transcription in the presence 
                     of cAMP."
     promoter        join(1571..1576,1577..1594,1595..1601)
                     /label=lac promoter
                     /note="promoter for the E. coli lac operon"
     protein_bind    1609..1625
                     /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     1614..1636
                     /label=M13/pUC Reverse
                     /note="In lacZ gene"
     primer_bind     1633..1649
                     /label=M13 rev
                     /note="common sequencing primer, one of multiple similar 
                     variants"
     primer_bind     1633..1649
                     /label=M13 Reverse
                     /note="In lacZ gene. Also called M13-rev"
     primer_bind     1668..1688
                     /label=T3
                     /note="T3 promoter, forward primer"
     promoter        1670..1688
                     /label=T3 promoter
                     /note="promoter for bacteriophage T3 RNA polymerase"
     promoter        1716..1942
                     /label=RSV promoter
                     /note="Rous sarcoma virus enhancer/promoter"
     LTR             1943..2123
                     /label=5' LTR (truncated)
                     /note="truncated 5' long terminal repeat (LTR) from HIV-1"
     misc_feature    2170..2295
                     /label=HIV-1 Psi
                     /note="packaging signal of human immunodeficiency virus 
                     type 1"
     misc_feature    2788..3021
                     /label=RRE
                     /note="The Rev response element (RRE) of HIV-1 allows for 
                     Rev-dependent mRNA export from the nucleus to the 
                     cytoplasm."
     CDS             3206..3250
                     /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        3481..3691
                     /label=SV40 promoter
                     /note="SV40 early promoter"
     promoter        3503..3699
                     /label=SV40 promoter
                     /note="SV40 early promoter"
     rep_origin      3550..3685
                     /label=SV40 ori
                     /note="SV40 origin of replication"
     CDS             join(3721..3723,3724..3726,3727..4443)
                     /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(4027..4046)
                     /label=EXFP-R
                     /note="For distinguishing EGFP variants, reverse primer"
     primer_bind     4374..4395
                     /label=EGFP-C
                     /note="EGFP, forward primer"
     misc_feature    4499..4616
                     /label=cPPT/CTS
                     /note="central polypurine tract and central termination 
                     sequence of HIV-1"
     promoter        4754..5932
                     /label=EF-1-alpha promoter
                     /note="strong constitutive promoter for human elongation 
                     factor EF-1-alpha"
     intron          4985..5923
                     /label=EF-1-alpha intron A
                     /note="intron upstream of the start codon of human 
                     EF-1-alpha"
     primer_bind     5880..5900
                     /label=EF1a-F
                     /note="Human elongation factor-1a promoter, forward primer"
     protein_bind    5952..5976
                     /gene="mutant version of attB"
                     /label=attB1
                     /bound_moiety="BP Clonase(TM)"
                     /note="recombination site for the Gateway(R) BP reaction"
     CDS             5988..6008
                     /codon_start=1
                     /product="nuclear localization signal of SV40 (simian virus
                     40) large T antigen"
                     /label=SV40 NLS
                     /translation="PKKKRKV"
     CDS             6015..10115
                     /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             6015..10115
                     /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             10122..10169
                     /codon_start=1
                     /product="bipartite nuclear localization signal from 
                     nucleoplasmin"
                     /label=nucleoplasmin NLS
                     /translation="KRPAATKKAGQAKKKK"
     CDS             10191..10247
                     /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             10254..10649
                     /codon_start=1
                     /gene="Aspergillus terreus BSD"
                     /product="blasticidin S deaminase"
                     /label=BSD
                     /note="confers resistance to blasticidin"
                     /translation="AKPLSQEESTLIERATATINSIPISEDYSVASAALSSDGRIFTGV
                     NVYHFTGGPCAELVVLGTAAAAAAGNLTCIVAIGNENRGILSPCGRCRQVLLDLHPGIK
                     AIVKDSDGQPTAVGIRELLPSGYVWEG"
     protein_bind    complement(10654..10678)
                     /gene="mutant version of attB"
                     /label=attB2
                     /bound_moiety="BP Clonase(TM)"
                     /note="recombination site for the Gateway(R) BP reaction"
     CDS             10686..10727
                     /codon_start=1
                     /product="epitope tag from simian virus 5"
                     /label=V5 tag
                     /translation="GKPIPNPLLGLDST"
     misc_feature    10778..11366
                     /label=WPRE
                     /note="woodchuck hepatitis virus posttranscriptional 
                     regulatory element"
     primer_bind     complement(10831..10851)
                     /label=WPRE-R
                     /note="WPRE, reverse primer"
     CDS             complement(11249..11260)
                     /codon_start=1
                     /product="Factor Xa recognition and cleavage site"
                     /label=Factor Xa site
                     /translation="IEGR"
     protein_bind    complement(11493..11526)
                     /label=loxP
                     /bound_moiety="Cre recombinase"
                     /note="Cre-mediated recombination occurs in the 8-bp core 
                     sequence (ATGTATGC) (Shaw et al., 2021)."
     LTR             11546..11726
                     /label=5' LTR (truncated)
                     /note="truncated 5' long terminal repeat (LTR) from HIV-1"
     polyA_signal    11803..11924
                     /label=SV40 poly(A) signal
                     /note="SV40 polyadenylation signal"
     primer_bind     complement(11840..11859)
                     /label=SV40pA-R
                     /note="SV40 polyA, reverse primer"
     primer_bind     11894..11913
                     /label=EBV-rev
                     /note="SV40 polyA terminator, reverse primer"
     rep_origin      11964..12099
                     /label=SV40 ori
                     /note="SV40 origin of replication"
     primer_bind     complement(12119..12138)
                     /label=T7
                     /note="T7 promoter, forward primer"
     promoter        complement(12120..12138)
                     /label=T7 promoter
                     /note="promoter for bacteriophage T7 RNA polymerase"
     primer_bind     complement(12148..12165)
                     /label=M13 Forward
                     /note="In lacZ gene. Also called M13-F20 or M13 (-21) 
                     Forward"
     primer_bind     complement(12148..12164)
                     /label=M13 fwd
                     /note="common sequencing primer, one of multiple similar 
                     variants"
     primer_bind     complement(12157..12179)
                     /label=M13/pUC Forward
                     /note="In lacZ gene"
     rep_origin      12306..12761
                     /direction=RIGHT
                     /label=f1 ori
                     /note="f1 bacteriophage origin of replication; arrow 
                     indicates direction of (+) strand synthesis"
     primer_bind     complement(12393..12412)
                     /label=F1ori-R
                     /note="F1 origin, reverse primer"
     primer_bind     12603..12624
                     /label=F1ori-F
                     /note="F1 origin, forward primer"
     promoter        12787..12891
                     /gene="bla"
                     /label=AmpR promoter
     CDS             join(12892..12960,12961..13264,1..488)
                     /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(13110..13129)
                     /label=Amp-R
                     /note="Ampicillin resistance gene, reverse primer"
ORIGIN
        1 taaccatgag tgataacact gcggccaact tacttctgac aacgatcgga ggaccgaagg
       61 agctaaccgc ttttttgcac aacatggggg atcatgtaac tcgccttgat cgttgggaac
      121 cggagctgaa tgaagccata ccaaacgacg agcgtgacac cacgatgcct gtagcaatgg
      181 caacaacgtt gcgcaaacta ttaactggcg aactacttac tctagcttcc cggcaacaat
      241 taatagactg gatggaggcg gataaagttg caggaccact tctgcgctcg gcccttccgg
      301 ctggctggtt tattgctgat aaatctggag ccggtgagcg tgggtctcgc ggtatcattg
      361 cagcactggg gccagatggt aagccctccc gtatcgtagt tatctacacg acggggagtc
      421 aggcaactat ggatgaacga aatagacaga tcgctgagat aggtgcctca ctgattaagc
      481 attggtaact gtcagaccaa gtttactcat atatacttta gattgattta aaacttcatt
      541 tttaatttaa aaggatctag gtgaagatcc tttttgataa tctcatgacc aaaatccctt
      601 aacgtgagtt ttcgttccac tgagcgtcag accccgtaga aaagatcaaa ggatcttctt
      661 gagatccttt ttttctgcgc gtaatctgct gcttgcaaac aaaaaaacca ccgctaccag
      721 cggtggtttg tttgccggat caagagctac caactctttt tccgaaggta actggcttca
      781 gcagagcgca gataccaaat actgttcttc tagtgtagcc gtagttaggc caccacttca
      841 agaactctgt agcaccgcct acatacctcg ctctgctaat cctgttacca gtggctgctg
      901 ccagtggcga taagtcgtgt cttaccgggt tggactcaag acgatagtta ccggataagg
      961 cgcagcggtc gggctgaacg gggggttcgt gcacacagcc cagcttggag cgaacgacct
     1021 acaccgaact gagataccta cagcgtgagc tatgagaaag cgccacgctt cccgaaggga
     1081 gaaaggcgga caggtatccg gtaagcggca gggtcggaac aggagagcgc acgagggagc
     1141 ttccaggggg aaacgcctgg tatctttata gtcctgtcgg gtttcgccac ctctgacttg
     1201 agcgtcgatt tttgtgatgc tcgtcagggg ggcggagcct atggaaaaac gccagcaacg
     1261 cggccttttt acggttcctg gccttttgct ggccttttgc tcacatgttc tttcctgcgt
     1321 tatcccctga ttctgtggat aaccgtatta ccgcctttga gtgagctgat accgctcgcc
     1381 gcagccgaac gaccgagcgc agcgagtcag tgagcgagga agcggaagag cgcccaatac
     1441 gcaaaccgcc tctccccgcg cgttggccga ttcattaatg cagctggcac gacaggtttc
     1501 ccgactggaa agcgggcagt gagcgcaacg caattaatgt gagttagctc actcattagg
     1561 caccccaggc tttacacttt atgcttccgg ctcgtatgtt gtgtggaatt gtgagcggat
     1621 aacaatttca cacaggaaac agctatgacc atgattacgc caagcgcgca attaaccctc
     1681 actaaaggga acaaaagctg gagctgcaag cttaatgtag tcttatgcaa tactcttgta
     1741 gtcttgcaac atggtaacga tgagttagca acatgcctta caaggagaga aaaagcaccg
     1801 tgcatgccga ttggtggaag taaggtggta cgatcgtgcc ttattaggaa ggcaacagac
     1861 gggtctgaca tggattggac gaaccactga attgccgcat tgcagagata ttgtatttaa
     1921 gtgcctagct cgatacataa acgggtctct ctggttagac cagatctgag cctgggagct
     1981 ctctggctaa ctagggaacc cactgcttaa gcctcaataa agcttgcctt gagtgcttca
     2041 agtagtgtgt gcccgtctgt tgtgtgactc tggtaactag agatccctca gaccctttta
     2101 gtcagtgtgg aaaatctcta gcagtggcgc ccgaacaggg acttgaaagc gaaagggaaa
     2161 ccagaggagc tctctcgacg caggactcgg cttgctgaag cgcgcacggc aagaggcgag
     2221 gggcggcgac tggtgagtac gccaaaaatt ttgactagcg gaggctagaa ggagagagat
     2281 gggtgcgaga gcgtcagtat taagcggggg agaattagat cgcgatggga aaaaattcgg
     2341 ttaaggccag ggggaaagaa aaaatataaa ttaaaacata tagtatgggc aagcagggag
     2401 ctagaacgat tcgcagttaa tcctggcctg ttagaaacat cagaaggctg tagacaaata
     2461 ctgggacagc tacaaccatc ccttcagaca ggatcagaag aacttagatc attatataat
     2521 acagtagcaa ccctctattg tgtgcatcaa aggatagaga taaaagacac caaggaagct
     2581 ttagacaaga tagaggaaga gcaaaacaaa agtaagacca ccgcacagca agcggccgct
     2641 gatcttcaga cctggaggag gagatatgag ggacaattgg agaagtgaat tatataaata
     2701 taaagtagta aaaattgaac cattaggagt agcacccacc aaggcaaaga gaagagtggt
     2761 gcagagagaa aaaagagcag tgggaatagg agctttgttc cttgggttct tgggagcagc
     2821 aggaagcact atgggcgcag cgtcaatgac gctgacggta caggccagac aattattgtc
     2881 tggtatagtg cagcagcaga acaatttgct gagggctatt gaggcgcaac agcatctgtt
     2941 gcaactcaca gtctggggca tcaagcagct ccaggcaaga atcctggctg tggaaagata
     3001 cctaaaggat caacagctcc tggggatttg gggttgctct ggaaaactca tttgcaccac
     3061 tgctgtgcct tggaatgcta gttggagtaa taaatctctg gaacagattt ggaatcacac
     3121 gacctggatg gagtgggaca gagaaattaa caattacaca agcttaatac actccttaat
     3181 tgaagaatcg caaaaccagc aagaaaagaa tgaacaagaa ttattggaat tagataaatg
     3241 ggcaagtttg tggaattggt ttaacataac aaattggctg tggtatataa aattattcat
     3301 aatgatagta ggaggcttgg taggtttaag aatagttttt gctgtacttt ctatagtgaa
     3361 tagagttagg cagggatatt caccattatc gtttcagacc cacctcccaa ccccgagggg
     3421 acccatgcat tgcatctcaa ttagtcagca accaggtgtg gaaagtcccc aggctcccca
     3481 gcaggcagaa gtatgcaaag catgcgtctc aattagtcag caaccatagt cccgccccta
     3541 actccgccca tcccgcccct aactccgccc agttccgccc attctccgcc ccatggctga
     3601 ctaatttttt ttatttatgc agaggccgag gccgcctcgg cctctgagct attccagaag
     3661 tagtgaggag gcttttttgg aggcctaggc ttttgcaaaa agctttctag aggtaccacc
     3721 atggtgagca agggcgagga gctgttcacc ggggtggtgc ccatcctggt cgagctggac
     3781 ggcgacgtaa acggccacaa gttcagcgtg tctggcgagg gcgagggcga tgccacctac
     3841 ggcaagctga ccctgaagtt catctgcacc accggcaagc tgcccgtgcc ctggcccacc
     3901 ctcgtgacca ccctgaccta cggcgtgcag tgcttcagcc gctaccccga ccacatgaag
     3961 cagcacgact tcttcaagtc cgccatgccc gaaggctacg tccaggagcg caccatcttc
     4021 ttcaaggacg acggcaacta caagacccgc gccgaggtga agttcgaggg cgacaccctg
     4081 gtgaaccgca tcgagctgaa gggcatcgac ttcaaggagg acggcaacat cctggggcac
     4141 aagctggagt acaactacaa cagccacaac gtctatatca tggccgacaa gcagaagaac
     4201 ggcatcaagg tgaacttcaa gatccgccac aacatcgagg acggcagcgt gcagctcgcc
     4261 gaccactacc agcagaacac ccccatcggc gacggccccg tgctgctgcc cgacaaccac
     4321 tacctgagca cccagtccgc cctgagcaaa gaccccaacg agaagcgcga tcacatggtc
     4381 ctgctggagt tcgtgaccgc cgccgggatc actctcggca tggacgagct gtacaagtcc
     4441 taaggcgcgc cgttaacgaa ttctagatct tgagacaaat ggcagtattc atccacaatt
     4501 ttaaaagaaa aggggggatt ggggggtaca gtgcagggga aagaatagta gacataatag
     4561 caacagacat acaaactaaa gaattacaaa aacaaattac aaaaattcaa aattttcggg
     4621 tttattacag ggacagcaga gatccacttt ggcgccggct cgaggcctgc aggtgcaaag
     4681 atggataaag ttttaaacag agaggaatct ttgcagctaa tggaccttct aggtcttgaa
     4741 aggagtggga attggctccg gtgcccgtca gtgggcagag cgcacatcgc ccacagtccc
     4801 cgagaagttg gggggagggg tcggcaattg aaccggtgcc tagagaaggt ggcgcggggt
     4861 aaactgggaa agtgatgtcg tgtactggct ccgccttttt cccgagggtg ggggagaacc
     4921 gtatataagt gcagtagtcg ccgtgaacgt tctttttcgc aacgggtttg ccgccagaac
     4981 acaggtaagt gccgtgtgtg gttcccgcgg gcctggcctc tttacgggtt atggcccttg
     5041 cgtgccttga attacttcca cctggctgca gtacgtgatt cttgatcccg agcttcgggt
     5101 tggaagtggg tgggagagtt cgaggccttg cgcttaagga gccccttcgc ctcgtgcttg
     5161 agttgaggcc tggcctgggc gctggggccg ccgcgtgcga atctggtggc accttcgcgc
     5221 ctgtctcgct gctttcgata agtctctagc catttaaaat ttttgatgac ctgctgcgac
     5281 gctttttttc tggcaagata gtcttgtaaa tgcgggccaa gatctgcaca ctggtatttc
     5341 ggtttttggg gccgcgggcg gcgacggggc ccgtgcgtcc cagcgcacat gttcggcgag
     5401 gcggggcctg cgagcgcggc caccgagaat cggacggggg tagtctcaag ctggccggcc
     5461 tgctctggtg cctggcctcg cgccgccgtg tatcgccccg ccctgggcgg caaggctggc
     5521 ccggtcggca ccagttgcgt gagcggaaag atggccgctt cccggccctg ctgcagggag
     5581 ctcaaaatgg aggacgcggc gctcgggaga gcgggcgggt gagtcaccca cacaaaggaa
     5641 aagggccttt ccgtcctcag ccgtcgcttc atgtgactcc acggagtacc gggcgccgtc
     5701 caggcacctc gattagttct cgagcttttg gagtacgtcg tctttaggtt ggggggaggg
     5761 gttttatgcg atggagtttc cccacactga gtgggtggag actgaagtta ggccagcttg
     5821 gcacttgatg taattctcct tggaatttgc cctttttgag tttggatctt ggttcattct
     5881 caagcctcag acagtggttc aaagtttttt tcttccattt caggtgtcgt gaggctagca
     5941 tcgattgatc aacaagtttg tacaaaaaag caggcttcac catggcacca aagaagaagc
     6001 ggaaggtcgc agccgacaag aagtacagca tcggcctgga catcggcacc aactctgtgg
     6061 gctgggccgt gatcaccgac gagtacaagg tgcccagcaa gaaattcaag gtgctgggca
     6121 acaccgaccg gcacagcatc aagaagaacc tgatcggagc cctgctgttc gacagcggcg
     6181 aaacagccga ggccacccgg ctgaagagaa ccgccagaag aagatacacc agacggaaga
     6241 accggatctg ctatctgcaa gagatcttca gcaacgagat ggccaaggtg gacgacagct
     6301 tcttccacag actggaagag tccttcctgg tggaagagga taagaagcac gagcggcacc
     6361 ccatcttcgg caacatcgtg gacgaggtgg cctaccacga gaagtacccc accatctacc
     6421 acctgagaaa gaaactggtg gacagcaccg acaaggccga cctgcggctg atctatctgg
     6481 ccctggccca catgatcaag ttccggggcc acttcctgat cgagggcgac ctgaaccccg
     6541 acaacagcga cgtggacaag ctgttcatcc agctggtgca gacctacaac cagctgttcg
     6601 aggaaaaccc catcaacgcc agcggcgtag acgccaaggc catcctgtct gccagactga
     6661 gcaagagcag acggctggaa aatctgatcg cccagctgcc cggcgagaag aagaatggcc
     6721 tgttcggaaa cctgattgcc ctgagcctgg gcctgacccc caacttcaag agcaacttcg
     6781 acctggccga ggatgccaaa ctgcagctga gcaaggacac ctacgacgac gacctggaca
     6841 acctgctggc ccagatcggc gaccagtacg ccgacctgtt tctggccgcc aagaacctgt
     6901 ccgacgccat cctgctgagc gacatcctga gagtgaacac cgagatcacc aaggcccccc
     6961 tgagcgcctc tatgatcaag agatacgacg agcaccacca ggacctgacc ctgctgaaag
     7021 ctctcgtgcg gcagcagctg cctgagaagt acaaagagat tttcttcgac cagagcaaga
     7081 acggctacgc cggctacatt gacggcggag ccagccagga agagttctac aagttcatca
     7141 agcccatcct ggaaaagatg gacggcaccg aggaactgct cgtgaagctg aacagagagg
     7201 acctgctgcg gaagcagcgt acgttcgaca acggcagcat cccccaccag atccacctgg
     7261 gagagctgca cgccattctg cggcggcagg aagattttta cccattcctg aaggacaacc
     7321 gggaaaagat cgagaagatc ctgaccttcc gcatccccta ctacgtgggc cctctggcca
     7381 ggggaaacag cagattcgcc tggatgacca gaaagagcga ggaaaccatc accccctgga
     7441 acttcgagga agtggtggac aagggcgcca gcgcccagag cttcatcgag cggatgacca
     7501 acttcgataa gaacctgccc aacgagaagg tgctgcccaa gcacagcctg ctgtacgagt
     7561 acttcaccgt gtataacgag ctgaccaaag tgaaatacgt gaccgaggga atgagaaagc
     7621 ccgccttcct gagcggcgag cagaaaaagg ccatcgtgga cctgctgttc aagaccaacc
     7681 ggaaagtgac cgtgaagcag ctgaaagagg actacttcaa gaaaatcgag tgcttcgact
     7741 ccgtggaaat ctccggcgtg gaagatcggt tcaacgcctc cctgggcaca taccacgatc
     7801 tgctgaaaat tatcaaggac aaggacttcc tggacaatga ggaaaacgag gacattctgg
     7861 aagatatcgt gctgaccctg acactgtttg aggacagaga gatgatcgag gaacggctga
     7921 aaacctatgc ccacctgttc gacgacaaag tgatgaagca gctgaagcgg cggagataca
     7981 ccggctgggg caggctgagc cggaagctga tcaacggcat ccgggacaag cagtccggca
     8041 agacaatcct ggatttcctg aagtccgacg gcttcgccaa cagaaacttc atgcagctga
     8101 tccacgacga cagcctgacc tttaaagagg acatccagaa agcccaggtg tccggccagg
     8161 gcgatagcct gcacgagcac attgccaatc tggccggcag ccccgccatt aagaagggca
     8221 tcctgcagac agtgaaggtg gtggacgagc tcgtgaaagt gatgggccgg cacaagcccg
     8281 agaacatcgt gatcgaaatg gccagagaga accagaccac ccagaaggga cagaagaaca
     8341 gccgcgagag aatgaagcgg atcgaagagg gcatcaaaga gctgggcagc cagatcctga
     8401 aagaacaccc cgtggaaaac acccagctgc agaacgagaa gctgtacctg tactacctgc
     8461 agaatgggcg ggatatgtac gtggaccagg aactggacat caaccggctg tccgactacg
     8521 atgtggacca tatcgtgcct cagagctttc tgaaggacga ctccatcgac aacaaggtgc
     8581 tgaccagaag cgacaagaac cggggcaaga gcgacaacgt gccctccgaa gaggtcgtga
     8641 agaagatgaa gaactactgg cggcagctgc tgaacgccaa gctgattacc cagagaaagt
     8701 tcgacaatct gaccaaggcc gagagaggcg gcctgagcga actggataag gccggcttca
     8761 tcaagagaca gctggtggaa acccggcaga tcacaaagca cgtggcacag atcctggact
     8821 cccggatgaa cactaagtac gacgagaatg acaagctgat ccgggaagtg aaagtgatca
     8881 ccctgaagtc caagctggtg tccgatttcc ggaaggattt ccagttttac aaagtgcgcg
     8941 agatcaacaa ctaccaccac gcccacgacg cctacctgaa cgccgtcgtg ggaaccgccc
     9001 tgatcaaaaa gtaccctaag ctggaaagcg agttcgtgta cggcgactac aaggtgtacg
     9061 acgtgcggaa gatgatcgcc aagagcgagc aggaaatcgg caaggctacc gccaagtact
     9121 tcttctacag caacatcatg aactttttca agaccgagat taccctggcc aacggcgaga
     9181 tccggaagcg gcctctgatc gagacaaacg gcgaaaccgg ggagatcgtg tgggataagg
     9241 gccgggattt tgccaccgtg cggaaagtgc tgagcatgcc ccaagtgaat atcgtgaaaa
     9301 agaccgaggt gcagacaggc ggcttcagca aagagtctat cctgcccaag aggaacagcg
     9361 ataagctgat cgccagaaag aaggactggg accctaagaa gtacggcggc ttcgacagcc
     9421 ccaccgtggc ctattctgtg ctggtggtgg ccaaagtgga aaagggcaag tccaagaaac
     9481 tgaagagtgt gaaagagctg ctggggatca ccatcatgga aagaagcagc ttcgagaaga
     9541 atcccatcga ctttctggaa gccaagggct acaaagaagt gaaaaaggac ctgatcatca
     9601 agctgcctaa gtactccctg ttcgagctgg aaaacggccg gaagagaatg ctggcctctg
     9661 ccggcgaact gcagaaggga aacgaactgg ccctgccctc caaatatgtg aacttcctgt
     9721 acctggccag ccactatgag aagctgaagg gctcccccga ggataatgag cagaaacagc
     9781 tgtttgtgga acagcacaag cactacctgg acgagatcat cgagcagatc agcgagttct
     9841 ccaagagagt gatcctggcc gacgctaatc tggacaaagt gctgtccgcc tacaacaagc
     9901 accgggataa gcccatcaga gagcaggccg agaatatcat ccacctgttt accctgacca
     9961 atctgggagc ccctgccgcc ttcaagtact ttgacaccac catcgaccgg aagaggtaca
    10021 ccagcaccaa agaggtgctg gacgccaccc tgatccacca gagcatcacc ggcctgtacg
    10081 agacacgaat cgatttgtct cagctgggag gcgacgccgc aaaaaggccg gcggccacga
    10141 aaaaggccgg ccaggcaaaa aagaaaaagg caggctcagg cggatccggc gcaacaaact
    10201 tctctctgct gaaacaagcc ggagatgtcg aagagaatcc tggaccgagc gctgccaagc
    10261 ctttgtctca agaagaatcc accctcattg aaagagcaac ggctacaatc aacagcatcc
    10321 ccatctctga agactacagc gtcgccagcg cagctctctc tagcgacggc cgcatcttca
    10381 ctggtgtcaa tgtatatcat tttactgggg gaccttgtgc agaactcgtg gtgctgggca
    10441 ctgctgctgc tgcggcagct ggcaacctga cttgtatcgt cgcgatcgga aatgagaaca
    10501 ggggcatctt gagcccctgc ggacggtgcc gacaggtgct tctcgatctg catcctggga
    10561 tcaaagccat agtgaaggac agtgatggac agccgacggc agttgggatt cgtgaattgc
    10621 tgccctctgg ttatgtgtgg gagggctgat aggacccagc tttcttgtac aaagtggttg
    10681 atatcggtaa gcctatccct aaccctctcc tcggtctcga ttctacgtag taatgaacta
    10741 gtaccggtta agtcgacaat caacgcgtta agtcgacaat caacctctgg attacaaaat
    10801 ttgtgaaaga ttgactggta ttcttaacta tgttgctcct tttacgctat gtggatacgc
    10861 tgctttaatg cctttgtatc atgctattgc ttcccgtatg gctttcattt tctcctcctt
    10921 gtataaatcc tggttgctgt ctctttatga ggagttgtgg cccgttgtca ggcaacgtgg
    10981 cgtggtgtgc actgtgtttg ctgacgcaac ccccactggt tggggcattg ccaccacctg
    11041 tcagctcctt tccgggactt tcgctttccc cctccctatt gccacggcgg aactcatcgc
    11101 cgcctgcctt gcccgctgct ggacaggggc tcggctgttg ggcactgaca attccgtggt
    11161 gttgtcgggg aaatcatcgt cctttccttg gctgctcgcc tgtgttgcca cctggattct
    11221 gcgcgggacg tccttctgct acgtcccttc ggccctcaat ccagcggacc ttccttcccg
    11281 cggcctgctg ccggctctgc ggcctcttcc gcgtcttcgc cttcgccctc agacgagtcg
    11341 gatctccctt tgggccgcct ccccgcgtcg actttaagac caatgactta caaggcagct
    11401 gtagatctta gccacttttt aaaagaaaag gggggactgg aagggctaat tcactcccaa
    11461 cgaagacaag atgggatcaa ttcaccatgg gaataacttc gtatagcata cattatacga
    11521 agttatgctg ctttttgctt gtactgggtc tctctggtta gaccagatct gagcctggga
    11581 gctctctggc taactaggga acccactgct taagcctcaa taaagcttgc cttgagtgct
    11641 tcaagtagtg tgtgcccgtc tgttgtgtga ctctggtaac tagagatccc tcagaccctt
    11701 ttagtcagtg tggaaaatct ctagcatacg tatagtagtt catgtcatct tattattcag
    11761 tatttataac ttgcaaagaa atgaatatca gagagtgaga ggaacttgtt tattgcagct
    11821 tataatggtt acaaataaag caatagcatc acaaatttca caaataaagc atttttttca
    11881 ctgcattcta gttgtggttt gtccaaactc atcaatgtat cttatcatgt ctggctctag
    11941 ctatcccgcc cctaactccg cccatcccgc ccctaactcc gcccagttcc gcccattctc
    12001 cgccccatgg ctgactaatt ttttttattt atgcagaggc cgaggccgcc tcggcctctg
    12061 agctattcca gaagtagtga ggaggctttt ttggaggcct agggacgtac ccaattcgcc
    12121 ctatagtgag tcgtattacg cgcgctcact ggccgtcgtt ttacaacgtc gtgactggga
    12181 aaaccctggc gttacccaac ttaatcgcct tgcagcacat ccccctttcg ccagctggcg
    12241 taatagcgaa gaggcccgca ccgatcgccc ttcccaacag ttgcgcagcc tgaatggcga
    12301 atgggacgcg ccctgtagcg gcgcattaag cgcggcgggt gtggtggtta cgcgcagcgt
    12361 gaccgctaca cttgccagcg ccctagcgcc cgctcctttc gctttcttcc cttcctttct
    12421 cgccacgttc gccggctttc cccgtcaagc tctaaatcgg gggctccctt tagggttccg
    12481 atttagtgct ttacggcacc tcgaccccaa aaaacttgat tagggtgatg gttcacgtag
    12541 tgggccatcg ccctgataga cggtttttcg ccctttgacg ttggagtcca cgttctttaa
    12601 tagtggactc ttgttccaaa ctggaacaac actcaaccct atctcggtct attcttttga
    12661 tttataaggg attttgccga tttcggccta ttggttaaaa aatgagctga tttaacaaaa
    12721 atttaacgcg aattttaaca aaatattaac gcttacaatt taggtggcac ttttcgggga
    12781 aatgtgcgcg gaacccctat ttgtttattt ttctaaatac attcaaatat gtatccgctc
    12841 atgagacaat aaccctgata aatgcttcaa taatattgaa aaaggaagag tatgagtatt
    12901 caacatttcc gtgtcgccct tattcccttt tttgcggcat tttgccttcc tgtttttgct
    12961 cacccagaaa cgctggtgaa agtaaaagat gctgaagatc agttgggtgc acgagtgggt
    13021 tacatcgaac tggatctcaa cagcggtaag atccttgaga gttttcgccc cgaagaacgt
    13081 tttccaatga tgagcacttt taaagttctg ctatgtggcg cggtattatc ccgtattgac
    13141 gccgggcaag agcaactcgg tcgccgcata cactattctc agaatgactt ggttgagtac
    13201 tcaccagtca cagaaaagca tcttacggat ggcatgacag taagagaatt atgcagtgct
    13261 gcca
//
