LOCUS       sequence-446064-       14888 bp    DNA     circular SYN 15-DEC-2025
DEFINITION  Entry vector to clone sgRNA(s) into the CROPseq-multi-v2 construct 
            with expression of SpCas9 and Puromycin resistance; v2 is compatible
            with T7 in vitro transcription detection.
ACCESSION   .
VERSION     .
KEYWORDS    .
SOURCE      synthetic DNA construct
  ORGANISM  synthetic DNA construct
REFERENCE   1  (bases 1 to 14888)
  AUTHORS   Walton RT, Qin Y, Blainey PC
  TITLE     CROPseq-multi: a versatile solution for multiplexed perturbation and
            decoding in pooled CRISPR screens
  JOURNAL   bioRxiv 2024.03.17.585235
REFERENCE   2  (bases 1 to 14888)
  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: "bioRxiv 
            2024.03.17.585235"
COMMENT     Sequence Label: CROPseq-multi-v2-SpCas9-P2A-Puro-BsmBI_entry
            (...
FEATURES             Location/Qualifiers
     source          1..14888
                     /mol_type="other DNA"
                     /organism="synthetic DNA construct"
     LTR             1..181
                     /label=5' LTR (truncated)
                     /note="truncated 5' long terminal repeat (LTR) from HIV-1"
     misc_feature    228..353
                     /label=HIV-1 Psi
                     /note="packaging signal of human immunodeficiency virus 
                     type 1"
     misc_feature    846..1079
                     /label=RRE
                     /note="The Rev response element (RRE) of HIV-1 allows for 
                     Rev-dependent mRNA export from the nucleus to the 
                     cytoplasm."
     CDS             1264..1308
                     /codon_start=1
                     /product="antigenic peptide corresponding to amino acids 
                     655 to 669 of the HIV envelope protein gp41 (Lutje Hulsik 
                     et al., 2013)"
                     /label=gp41 peptide
                     /note="recognized by the 2H10 single-chain llama nanobody"
                     /translation="KNEQELLELDKWASL"
     misc_feature    1489..1606
                     /label=cPPT/CTS
                     /note="central polypurine tract and central termination 
                     sequence of HIV-1"
     promoter        1745..2923
                     /label=EF-1-alpha promoter
                     /note="strong constitutive promoter for human elongation 
                     factor EF-1-alpha"
     intron          1976..2914
                     /label=EF-1-alpha intron A
                     /note="intron upstream of the start codon of human 
                     EF-1-alpha"
     primer_bind     2871..2891
                     /label=EF1a-F
                     /note="Human elongation factor-1a promoter, forward primer"
     regulatory      2930..2939
                     /label=Kozak sequence
                     /note="vertebrate consensus sequence for strong initiation 
                     of translation (Kozak, 1987)"
                     /regulatory_class="other"
     CDS             2936..7039
                     /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="MDKKYSIGLDIGTNSVGWAVITDEYKVPSKKFKVLGNTDRHSIKK
                     NLIGALLFDSGETAEATRLKRTARRRYTRRKNRICYLQEIFSNEMAKVDDSFFHRLEES
                     FLVEEDKKHERHPIFGNIVDEVAYHEKYPTIYHLRKKLVDSTDKADLRLIYLALAHMIK
                     FRGHFLIEGDLNPDNSDVDKLFIQLVQTYNQLFEENPINASGVDAKAILSARLSKSRRL
                     ENLIAQLPGEKKNGLFGNLIALSLGLTPNFKSNFDLAEDAKLQLSKDTYDDDLDNLLAQ
                     IGDQYADLFLAAKNLSDAILLSDILRVNTEITKAPLSASMIKRYDEHHQDLTLLKALVR
                     QQLPEKYKEIFFDQSKNGYAGYIDGGASQEEFYKFIKPILEKMDGTEELLVKLNREDLL
                     RKQRTFDNGSIPHQIHLGELHAILRRQEDFYPFLKDNREKIEKILTFRIPYYVGPLARG
                     NSRFAWMTRKSEETITPWNFEEVVDKGASAQSFIERMTNFDKNLPNEKVLPKHSLLYEY
                     FTVYNELTKVKYVTEGMRKPAFLSGEQKKAIVDLLFKTNRKVTVKQLKEDYFKKIECFD
                     SVEISGVEDRFNASLGTYHDLLKIIKDKDFLDNEENEDILEDIVLTLTLFEDREMIEER
                     LKTYAHLFDDKVMKQLKRRRYTGWGRLSRKLINGIRDKQSGKTILDFLKSDGFANRNFM
                     QLIHDDSLTFKEDIQKAQVSGQGDSLHEHIANLAGSPAIKKGILQTVKVVDELVKVMGR
                     HKPENIVIEMARENQTTQKGQKNSRERMKRIEEGIKELGSQILKEHPVENTQLQNEKLY
                     LYYLQNGRDMYVDQELDINRLSDYDVDHIVPQSFLKDDSIDNKVLTRSDKNRGKSDNVP
                     SEEVVKKMKNYWRQLLNAKLITQRKFDNLTKAERGGLSELDKAGFIKRQLVETRQITKH
                     VAQILDSRMNTKYDENDKLIREVKVITLKSKLVSDFRKDFQFYKVREINNYHHAHDAYL
                     NAVVGTALIKKYPKLESEFVYGDYKVYDVRKMIAKSEQEIGKATAKYFFYSNIMNFFKT
                     EITLANGEIRKRPLIETNGETGEIVWDKGRDFATVRKVLSMPQVNIVKKTEVQTGGFSK
                     ESILPKRNSDKLIARKKDWDPKKYGGFDSPTVAYSVLVVAKVEKGKSKKLKSVKELLGI
                     TIMERSSFEKNPIDFLEAKGYKEVKKDLIIKLPKYSLFELENGRKRMLASAGELQKGNE
                     LALPSKYVNFLYLASHYEKLKGSPEDNEQKQLFVEQHKHYLDEIIEQISEFSKRVILAD
                     ANLDKVLSAYNKHRDKPIREQAENIIHLFTLTNLGAPAAFKYFDTTIDRKRYTSTKEVL
                     DATLIHQSITGLYETRIDLSQLGGD"
     CDS             2936..7039
                     /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="MDKKYSIGLDIGTNSVGWAVITDEYKVPSKKFKVLGNTDRHSIKK
                     NLIGALLFDSGETAEATRLKRTARRRYTRRKNRICYLQEIFSNEMAKVDDSFFHRLEES
                     FLVEEDKKHERHPIFGNIVDEVAYHEKYPTIYHLRKKLVDSTDKADLRLIYLALAHMIK
                     FRGHFLIEGDLNPDNSDVDKLFIQLVQTYNQLFEENPINASGVDAKAILSARLSKSRRL
                     ENLIAQLPGEKKNGLFGNLIALSLGLTPNFKSNFDLAEDAKLQLSKDTYDDDLDNLLAQ
                     IGDQYADLFLAAKNLSDAILLSDILRVNTEITKAPLSASMIKRYDEHHQDLTLLKALVR
                     QQLPEKYKEIFFDQSKNGYAGYIDGGASQEEFYKFIKPILEKMDGTEELLVKLNREDLL
                     RKQRTFDNGSIPHQIHLGELHAILRRQEDFYPFLKDNREKIEKILTFRIPYYVGPLARG
                     NSRFAWMTRKSEETITPWNFEEVVDKGASAQSFIERMTNFDKNLPNEKVLPKHSLLYEY
                     FTVYNELTKVKYVTEGMRKPAFLSGEQKKAIVDLLFKTNRKVTVKQLKEDYFKKIECFD
                     SVEISGVEDRFNASLGTYHDLLKIIKDKDFLDNEENEDILEDIVLTLTLFEDREMIEER
                     LKTYAHLFDDKVMKQLKRRRYTGWGRLSRKLINGIRDKQSGKTILDFLKSDGFANRNFM
                     QLIHDDSLTFKEDIQKAQVSGQGDSLHEHIANLAGSPAIKKGILQTVKVVDELVKVMGR
                     HKPENIVIEMARENQTTQKGQKNSRERMKRIEEGIKELGSQILKEHPVENTQLQNEKLY
                     LYYLQNGRDMYVDQELDINRLSDYDVDHIVPQSFLKDDSIDNKVLTRSDKNRGKSDNVP
                     SEEVVKKMKNYWRQLLNAKLITQRKFDNLTKAERGGLSELDKAGFIKRQLVETRQITKH
                     VAQILDSRMNTKYDENDKLIREVKVITLKSKLVSDFRKDFQFYKVREINNYHHAHDAYL
                     NAVVGTALIKKYPKLESEFVYGDYKVYDVRKMIAKSEQEIGKATAKYFFYSNIMNFFKT
                     EITLANGEIRKRPLIETNGETGEIVWDKGRDFATVRKVLSMPQVNIVKKTEVQTGGFSK
                     ESILPKRNSDKLIARKKDWDPKKYGGFDSPTVAYSVLVVAKVEKGKSKKLKSVKELLGI
                     TIMERSSFEKNPIDFLEAKGYKEVKKDLIIKLPKYSLFELENGRKRMLASAGELQKGNE
                     LALPSKYVNFLYLASHYEKLKGSPEDNEQKQLFVEQHKHYLDEIIEQISEFSKRVILAD
                     ANLDKVLSAYNKHRDKPIREQAENIIHLFTLTNLGAPAAFKYFDTTIDRKRYTSTKEVL
                     DATLIHQSITGLYETRIDLSQLGGD"
     CDS             7040..7087
                     /codon_start=1
                     /product="bipartite nuclear localization signal from 
                     nucleoplasmin"
                     /label=nucleoplasmin NLS
                     /translation="KRPAATKKAGQAKKKK"
     CDS             7088..7111
                     /codon_start=1
                     /product="FLAG(R) epitope tag, followed by an enterokinase 
                     cleavage site"
                     /label=FLAG
                     /translation="DYKDDDDK"
     CDS             7121..7177
                     /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             7178..7777
                     /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(7178..7197)
                     /label=Puro-R
                     /note="Puromycin resistance gene, reverse primer. Also 
                     called puro-variant-R"
     primer_bind     7674..7694
                     /label=Puro-F
                     /note="Puromycin resistance gene, forward primer"
     misc_feature    7793..8381
                     /label=WPRE
                     /note="woodchuck hepatitis virus posttranscriptional 
                     regulatory element"
     primer_bind     complement(7846..7866)
                     /label=WPRE-R
                     /note="WPRE, reverse primer"
     CDS             complement(8264..8275)
                     /codon_start=1
                     /product="Factor Xa recognition and cleavage site"
                     /label=Factor Xa site
                     /translation="IEGR"
     promoter        8516..8534
                     /label=T7 promoter
                     /note="promoter for bacteriophage T7 RNA polymerase"
     promoter        complement(10562..10810)
                     /label=U6 promoter
                     /note="RNA polymerase III promoter for human U6 snRNA 
                     (Domitrovich & Kunkel, 2003)"
     primer_bind     complement(10620..10639)
                     /label=LKO.1 5'
                     /note="Human U6 promoter, forward primer"
     primer_bind     complement(10790..10810)
                     /label=hU6-F
                     /note="Human U6 promoter, forward primer"
     LTR             10857..11037
                     /label=5' LTR (truncated)
                     /note="truncated 5' long terminal repeat (LTR) from HIV-1"
     polyA_signal    11115..11236
                     /label=SV40 poly(A) signal
                     /note="SV40 polyadenylation signal"
     primer_bind     complement(11152..11171)
                     /label=SV40pA-R
                     /note="SV40 polyA, reverse primer"
     primer_bind     11206..11225
                     /label=EBV-rev
                     /note="SV40 polyA terminator, reverse primer"
     rep_origin      11276..11411
                     /label=SV40 ori
                     /note="SV40 origin of replication"
     promoter        complement(11432..11450)
                     /label=T7 promoter
                     /note="promoter for bacteriophage T7 RNA polymerase"
     primer_bind     complement(11460..11477)
                     /label=M13 Forward
                     /note="In lacZ gene. Also called M13-F20 or M13 (-21) 
                     Forward"
     primer_bind     complement(11460..11476)
                     /label=M13 fwd
                     /note="common sequencing primer, one of multiple similar 
                     variants"
     primer_bind     complement(11469..11491)
                     /label=M13/pUC Forward
                     /note="In lacZ gene"
     rep_origin      11618..12073
                     /direction=RIGHT
                     /label=f1 ori
                     /note="f1 bacteriophage origin of replication; arrow 
                     indicates direction of (+) strand synthesis"
     primer_bind     complement(11705..11724)
                     /label=F1ori-R
                     /note="F1 origin, reverse primer"
     primer_bind     11915..11936
                     /label=F1ori-F
                     /note="F1 origin, forward primer"
     promoter        12099..12203
                     /gene="bla"
                     /label=AmpR promoter
     CDS             join(12204..12272,12273..13064)
                     /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(12422..12441)
                     /label=Amp-R
                     /note="Ampicillin resistance gene, reverse primer"
     rep_origin      13235..13823
                     /direction=RIGHT
                     /label=ori
                     /note="high-copy-number ColE1/pMB1/pBR322/pUC origin of 
                     replication"
     primer_bind     13724..13743
                     /label=pBR322ori-F
                     /note="pBR322 origin, forward primer"
     primer_bind     13977..13994
                     /label=L4440
                     /note="L4440 vector, forward primer"
     protein_bind    14111..14132
                     /label=CAP binding site
                     /bound_moiety="E. coli catabolite activator protein"
                     /note="CAP binding activates transcription in the presence 
                     of cAMP."
     promoter        join(14147..14152,14153..14170,14171..14177)
                     /label=lac promoter
                     /note="promoter for the E. coli lac operon"
     protein_bind    14185..14201
                     /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     14190..14212
                     /label=M13/pUC Reverse
                     /note="In lacZ gene"
     primer_bind     14209..14225
                     /label=M13 rev
                     /note="common sequencing primer, one of multiple similar 
                     variants"
     primer_bind     14209..14225
                     /label=M13 Reverse
                     /note="In lacZ gene. Also called M13-rev"
     primer_bind     14244..14264
                     /label=T3
                     /note="T3 promoter, forward primer"
     promoter        14246..14264
                     /label=T3 promoter
                     /note="promoter for bacteriophage T3 RNA polymerase"
     enhancer        14292..14671
                     /label=CMV enhancer
                     /note="human cytomegalovirus immediate early enhancer"
     promoter        14673..14871
                     /label=CMV promoter
                     /note="human cytomegalovirus (CMV) immediate early 
                     promoter"
     primer_bind     14826..14846
                     /label=CMV-F
                     /note="Human CMV immediate early promoter, forward primer"
ORIGIN
        1 gggtctctct ggttagacca gatctgagcc tgggagctct ctggctaact agggaaccca
       61 ctgcttaagc ctcaataaag cttgccttga gtgcttcaag tagtgtgtgc ccgtctgttg
      121 tgtgactctg gtaactagag atccctcaga cccttttagt cagtgtggaa aatctctagc
      181 agtggcgccc gaacagggac ttgaaagcga aagggaaacc agaggagctc tctcgacgca
      241 ggactcggct tgctgaagcg cgcacggcaa gaggcgaggg gcggcgactg gtgagtacgc
      301 caaaaatttt gactagcgga ggctagaagg agagagatgg gtgcgagagc gtcagtatta
      361 agcgggggag aattagatcg cgatgggaaa aaattcggtt aaggccaggg ggaaagaaaa
      421 aatataaatt aaaacatata gtatgggcaa gcagggagct agaacgattc gcagttaatc
      481 ctggcctgtt agaaacatca gaaggctgta gacaaatact gggacagcta caaccatccc
      541 ttcagacagg atcagaagaa cttagatcat tatataatac agtagcaacc ctctattgtg
      601 tgcatcaaag gatagagata aaagacacca aggaagcttt agacaagata gaggaagagc
      661 aaaacaaaag taagaccacc gcacagcaag cggccgctga tcttcagacc tggaggagga
      721 gatatgaggg acaattggag aagtgaatta tataaatata aagtagtaaa aattgaacca
      781 ttaggagtag cacccaccaa ggcaaagaga agagtggtgc agagagaaaa aagagcagtg
      841 ggaataggag ctttgttcct tgggttcttg ggagcagcag gaagcactat gggcgcagcg
      901 tcaatgacgc tgacggtaca ggccagacaa ttattgtctg gtatagtgca gcagcagaac
      961 aatttgctga gggctattga ggcgcaacag catctgttgc aactcacagt ctggggcatc
     1021 aagcagctcc aggcaagaat cctggctgtg gaaagatacc taaaggatca acagctcctg
     1081 gggatttggg gttgctctgg aaaactcatt tgcaccactg ctgtgccttg gaatgctagt
     1141 tggagtaata aatctctgga acagatttgg aatcacacga cctggatgga gtgggacaga
     1201 gaaattaaca attacacaag cttaatacac tccttaattg aagaatcgca aaaccagcaa
     1261 gaaaagaatg aacaagaatt attggaatta gataaatggg caagtttgtg gaattggttt
     1321 aacataacaa attggctgtg gtatataaaa ttattcataa tgatagtagg aggcttggta
     1381 ggtttaagaa tagtttttgc tgtactttct atagtgaata gagttaggca gggatattca
     1441 ccattatcgt ttcagaatct tgagacaaat ggcagtattc atccacaatt ttaaaagaaa
     1501 aggggggatt ggggggtaca gtgcagggga aagaatagta gacataatag caacagacat
     1561 acaaactaaa gaattacaaa aacaaattac aaaaattcaa aattttcggg tttattacag
     1621 ggacagcaga gatccacttt ggcgccggct cgagggggcc cgggtgcaaa gatggataaa
     1681 gttttaaaca gagaggaatc tttgcagcta atggaccttc taggtcttga aaggagtggg
     1741 aattggctcc ggtgcccgtc agtgggcaga gcgcacatcg cccacagtcc ccgagaagtt
     1801 ggggggaggg gtcggcaatt gatccggtgc ctagagaagg tggcgcgggg taaactggga
     1861 aagtgatgtc gtgtactggc tccgcctttt tcccgagggt gggggagaac cgtatataag
     1921 tgcagtagtc gccgtgaacg ttctttttcg caacgggttt gccgccagaa cacaggtaag
     1981 tgccgtgtgt ggttcccgcg ggcctggcct ctttacgggt tatggccctt gcgtgccttg
     2041 aattacttcc acctggctgc agtacgtgat tcttgatccc gagcttcggg ttggaagtgg
     2101 gtgggagagt tcgaggcctt gcgcttaagg agccccttcg cctcgtgctt gagttgaggc
     2161 ctggcctggg cgctggggcc gccgcgtgcg aatctggtgg caccttcgcg cctgtctcgc
     2221 tgctttcgat aagtctctag ccatttaaaa tttttgatga cctgctgcga cgcttttttt
     2281 ctggcaagat agtcttgtaa atgcgggcca agatctgcac actggtattt cggtttttgg
     2341 ggccgcgggc ggcgacgggg cccgtgcgtc ccagcgcaca tgttcggcga ggcggggcct
     2401 gcgagcgcgg ccaccgagaa tcggacgggg gtagtctcaa gctggccggc ctgctctggt
     2461 gcctggcctc gcgccgccgt gtatcgcccc gccctgggcg gcaaggctgg cccggtcggc
     2521 accagttgcg tgagcggaaa gatggccgct tcccggccct gctgcaggga gctcaaaatg
     2581 gaggacgcgg cgctcgggag agcgggcggg tgagtcaccc acacaaagga aaagggcctt
     2641 tccgtcctca gccgtcgctt catgtgactc cacggagtac cgggcgccgt ccaggcacct
     2701 cgattagttc tcgagctttt ggagtacgtc gtctttaggt tggggggagg ggttttatgc
     2761 gatggagttt ccccacactg agtgggtgga gactgaagtt aggccagctt ggcacttgat
     2821 gtaattctcc ttggaatttg ccctttttga gtttggatct tggttcattc tcaagcctca
     2881 gacagtggtt caaagttttt ttcttccatt tcaggtgtcg tgacgtacgg ccaccatgga
     2941 caagaagtac agcatcggcc tggacatcgg caccaactct gtgggctggg ccgtgatcac
     3001 cgacgagtac aaggtgccca gcaagaaatt caaggtgctg ggcaacaccg accggcacag
     3061 catcaagaag aacctgatcg gagccctgct gttcgacagc ggcgaaacag ccgaggccac
     3121 ccggctgaag agaaccgcca gaagaagata caccagacgg aagaaccgga tctgctatct
     3181 gcaagagatc ttcagcaacg agatggccaa ggtggacgac agcttcttcc acagactgga
     3241 agagtccttc ctggtggaag aggataagaa gcacgagcgg caccccatct tcggcaacat
     3301 cgtggacgag gtggcctacc acgagaagta ccccaccatc taccacctga gaaagaaact
     3361 ggtggacagc accgacaagg ccgacctgcg gctgatctat ctggccctgg cccacatgat
     3421 caagttccgg ggccacttcc tgatcgaggg cgacctgaac cccgacaaca gcgacgtgga
     3481 caagctgttc atccagctgg tgcagaccta caaccagctg ttcgaggaaa accccatcaa
     3541 cgccagcggc gtggacgcca aggccatcct gtctgccaga ctgagcaaga gcagacggct
     3601 ggaaaatctg atcgcccagc tgcccggcga gaagaagaat ggcctgttcg gaaacctgat
     3661 tgccctgagc ctgggcctga cccccaactt caagagcaac ttcgacctgg ccgaggatgc
     3721 caaactgcag ctgagcaagg acacctacga cgacgacctg gacaacctgc tggcccagat
     3781 cggcgaccag tacgccgacc tgtttctggc cgccaagaac ctgtccgacg ccatcctgct
     3841 gagcgacatc ctgagagtga acaccgagat caccaaggcc cccctgagcg cctctatgat
     3901 caagagatac gacgagcacc accaggacct gaccctgctg aaagctctcg tgcggcagca
     3961 gctgcctgag aagtacaaag agattttctt cgaccagagc aagaacggct acgccggcta
     4021 cattgacggc ggagccagcc aggaagagtt ctacaagttc atcaagccca tcctggaaaa
     4081 gatggacggc accgaggaac tgctcgtgaa gctgaacaga gaggacctgc tgcggaagca
     4141 gcggaccttc gacaacggca gcatccccca ccagatccac ctgggagagc tgcacgccat
     4201 tctgcggcgg caggaagatt tttacccatt cctgaaggac aaccgggaaa agatcgagaa
     4261 gatcctgacc ttccgcatcc cctactacgt gggccctctg gccaggggaa acagcagatt
     4321 cgcctggatg accagaaaga gcgaggaaac catcaccccc tggaacttcg aggaagtggt
     4381 ggacaagggc gcttccgccc agagcttcat cgagcggatg accaacttcg ataagaacct
     4441 gcccaacgag aaggtgctgc ccaagcacag cctgctgtac gagtacttca ccgtgtataa
     4501 cgagctgacc aaagtgaaat acgtgaccga gggaatgaga aagcccgcct tcctgagcgg
     4561 cgagcagaaa aaggccatcg tggacctgct gttcaagacc aaccggaaag tgaccgtgaa
     4621 gcagctgaaa gaggactact tcaagaaaat cgagtgcttc gactccgtgg aaatctccgg
     4681 cgtggaagat cggttcaacg cctccctggg cacataccac gatctgctga aaattatcaa
     4741 ggacaaggac ttcctggaca atgaggaaaa cgaggacatt ctggaagata tcgtgctgac
     4801 cctgacactg tttgaggaca gagagatgat cgaggaacgg ctgaaaacct atgcccacct
     4861 gttcgacgac aaagtgatga agcagctgaa gcggcggaga tacaccggct ggggcaggct
     4921 gagccggaag ctgatcaacg gcatccggga caagcagtcc ggcaagacaa tcctggattt
     4981 cctgaagtcc gacggcttcg ccaacagaaa cttcatgcag ctgatccacg acgacagcct
     5041 gacctttaaa gaggacatcc agaaagccca ggtgtccggc cagggcgata gcctgcacga
     5101 gcacattgcc aatctggccg gcagccccgc cattaagaag ggcatcctgc agacagtgaa
     5161 ggtggtggac gagctcgtga aagtgatggg ccggcacaag cccgagaaca tcgtgatcga
     5221 aatggccaga gagaaccaga ccacccagaa gggacagaag aacagccgcg agagaatgaa
     5281 gcggatcgaa gagggcatca aagagctggg cagccagatc ctgaaagaac accccgtgga
     5341 aaacacccag ctgcagaacg agaagctgta cctgtactac ctgcagaatg ggcgggatat
     5401 gtacgtggac caggaactgg acatcaaccg gctgtccgac tacgatgtgg accatatcgt
     5461 gcctcagagc tttctgaagg acgactccat cgacaacaag gtgctgacca gaagcgacaa
     5521 gaaccggggc aagagcgaca acgtgccctc cgaagaggtc gtgaagaaga tgaagaacta
     5581 ctggcggcag ctgctgaacg ccaagctgat tacccagaga aagttcgaca atctgaccaa
     5641 ggccgagaga ggcggcctga gcgaactgga taaggccggc ttcatcaaga gacagctggt
     5701 ggaaacccgg cagatcacaa agcacgtggc acagatcctg gactcccgga tgaacactaa
     5761 gtacgacgag aatgacaagc tgatccggga agtgaaagtg atcaccctga agtccaagct
     5821 ggtgtccgat ttccggaagg atttccagtt ttacaaagtg cgcgagatca acaactacca
     5881 ccacgcccac gacgcctacc tgaacgccgt cgtgggaacc gccctgatca aaaagtaccc
     5941 taagctggaa agcgagttcg tgtacggcga ctacaaggtg tacgacgtgc ggaagatgat
     6001 cgccaagagc gagcaggaaa tcggcaaggc taccgccaag tacttcttct acagcaacat
     6061 catgaacttt ttcaagaccg agattaccct ggccaacggc gagatccgga agcggcctct
     6121 gatcgagaca aacggcgaaa ccggggagat cgtgtgggat aagggccggg attttgccac
     6181 cgtgcggaaa gtgctgagca tgccccaagt gaatatcgtg aaaaagaccg aggtgcagac
     6241 aggcggcttc agcaaagagt ctatcctgcc caagaggaac agcgataagc tgatcgccag
     6301 aaagaaggac tgggacccta agaagtacgg cggcttcgac agccccaccg tggcctattc
     6361 tgtgctggtg gtggccaaag tggaaaaggg caagtccaag aaactgaaga gtgtgaaaga
     6421 gctactgggg atcaccatca tggaaagaag cagcttcgag aagaatccca tcgactttct
     6481 ggaagccaag ggctacaaag aagtgaaaaa ggacctgatc atcaagctgc ctaagtactc
     6541 cctgttcgag ctggaaaacg gccggaagag aatgctggcc tctgccggcg aactgcagaa
     6601 gggaaacgaa ctggccctgc cctccaaata tgtgaacttc ctgtacctgg ccagccacta
     6661 tgagaagctg aagggctccc ccgaggataa tgagcagaaa cagctgtttg tggaacagca
     6721 caagcactac ctggacgaga tcatcgagca gatcagcgag ttctccaaga gagtgatcct
     6781 ggccgacgct aatctggaca aagtgctgtc cgcctacaac aagcaccggg ataagcccat
     6841 cagagagcag gccgagaata tcatccacct gtttaccctg accaatctgg gagcccctgc
     6901 cgccttcaag tactttgaca ccaccatcga ccggaagagg tacaccagca ccaaagaggt
     6961 gctggacgcc accctgatcc accagagcat caccggcctg tacgagacac ggatcgacct
     7021 gtctcagctg ggaggcgaca agcgacctgc cgccacaaag aaggctggac aggctaagaa
     7081 gaagaaagat tacaaagacg atgacgataa gggatccggc gcaacaaact tctctctgct
     7141 gaaacaagcc ggagatgtcg aagagaatcc tggaccgatg accgagtaca agcccacggt
     7201 gcgcctcgcc acccgcgacg acgtccccag ggccgtacgc accctcgccg ccgcgttcgc
     7261 cgactacccc gccacgcgcc acaccgtcga tccggaccgc cacatcgagc gggtcaccga
     7321 gctgcaagaa ctcttcctca cgcgcgtcgg gctcgacatc ggcaaggtgt gggtcgcgga
     7381 cgacggcgcc gccgtggcgg tctggaccac gccggagagc gtcgaagcgg gggcggtgtt
     7441 cgccgagatc ggcccgcgca tggccgagtt gagcggttcc cggctggccg cgcagcaaca
     7501 gatggaaggc ctcctggcgc cgcaccggcc caaggagccc gcgtggttcc tggccaccgt
     7561 cggagtctcg cccgaccacc agggcaaggg tctgggcagc gccgtcgtgc tccccggagt
     7621 ggaggcggcc gagcgcgccg gggtgcccgc cttcctggag acctccgcgc cccgcaacct
     7681 ccccttctac gagcggctcg gcttcaccgt caccgccgac gtcgaggtgc ccgaaggacc
     7741 gcgcacctgg tgcatgaccc gcaagcccgg tgcctgaacg cgttaagtcg acaatcaacc
     7801 tctggattac aaaatttgtg aaagattgac tggtattctt aactatgttg ctccttttac
     7861 gctatgtgga tacgctgctt taatgccttt gtatcatgct attgcttccc gtatggcttt
     7921 cattttctcc tccttgtata aatcctggtt gctgtctctt tatgaggagt tgtggcccgt
     7981 tgtcaggcaa cgtggcgtgg tgtgcactgt gtttgctgac gcaaccccca ctggttgggg
     8041 cattgccacc acctgtcagc tcctttccgg gactttcgct ttccccctcc ctattgccac
     8101 ggcggaactc atcgccgcct gccttgcccg ctgctggaca ggggctcggc tgttgggcac
     8161 tgacaattcc gtggtgttgt cggggaaatc atcgtccttt ccttggctgc tcgcctgtgt
     8221 tgccacctgg attctgcgcg ggacgtcctt ctgctacgtc ccttcggccc tcaatccagc
     8281 ggaccttcct tcccgcggcc tgctgccggc tctgcggcct cttccgcgtg ttcgccttcg
     8341 ccctcagacg agtcggatct ccctttgggc cgcctccccg cgtcgacttt aagaccaatg
     8401 acttacaagg cagctgtaga tcttagccac tttttaaaag aaaagggggg actggaaggg
     8461 ctaattcact cccaacgaag agaagatttc accattatcg tttcagaccc atttttaata
     8521 cgactcacta tagggaaaaa aagcaccgac tcggtgccac tttttcaagt tgataacgga
     8581 ctagccttat ttcaacttgc tatgctgtta gagacgtaca aaaaagagca agaagctaaa
     8641 aaagatttaa aaattatttt tagcgcagtt aatggaacag gaactaaatt taccccaaaa
     8701 atattacgtg aatcaggata taacgttatt gaggttgaag agcatgcatt tgaagatgaa
     8761 acatttaaaa atgttgtaaa tccaaatcca gaatttgatc ctgcatgaaa aataccgctt
     8821 gaatatggta ttaaacatga tgcagatatt attattatga atgacccaga tgctgacaga
     8881 tttggaatgg caataaaaca tgatggtcat tttgtaagat tagatggaaa tcaaacagga
     8941 ccaattttaa ttgattgaaa attatcaaat ctaaaacgct taaatagcat tccaaaaaat
     9001 ccggctctat attcaagttt tgtaacaagt gatttgggtg atagaatcgc tcatgaaaaa
     9061 tatggagtta atattgtaaa aactttaact ggatttaaat gaatgggtag agaaattgct
     9121 aaagaagaag ataacggatt aaattttgtt tttgcttatg aagaaagtta tggatatgta
     9181 attgatgact cagctagaga taaagatgga atacaagctt ctatattaat agcagaggct
     9241 gcttgatttt ataaaaaaca aaataaaaca ttagtagact atttagaaga tttatttaaa
     9301 gaaatgggtg catattacac tttcacttta aacttgaatt ttaaaccaga agaaaagaaa
     9361 ttaaaaattg aaccattaat gaaatcattg agagcaacac ccttaactca aattgctgga
     9421 cttaaagttg ttaatgttga agactacatc gatggaatgt ataatatgcc aggacaagac
     9481 ttactaaaat tttatttaga agataagtca tgatttgctg ttcgcccaag tggaactgaa
     9541 cctaaactaa aaatttattt tataggtgtt ggtgaatctg ttcaaaacgc taaagttaaa
     9601 gtagacgaaa ttattaaaga attaaaatta aaaatgaata tataggagaa aaaatgaaac
     9661 taaacaaata tatagatcac acattattaa aacaagatgc tacgaaagct gaaattaaac
     9721 aattatgtga tgaagcaatt gaatttgatt ttgcaacagt ttgtgttaat tcatattgaa
     9781 caagctattg taaagaatta ttaaaaggca caaatgtagg aataacaaat gttgtaggtt
     9841 ttcctctagg tgcatgcaca acagctacaa aagcattcga agtttctgaa gcaattaaag
     9901 atggtgcaac agaaattgat atggtattaa atattggtgc attaaaagac aaaaattatg
     9961 aattagtttt agaagacatg aaagctgtaa aaaaagcagc tggatcacat gttgttaaat
    10021 gtattatgga aaattgttta ttaacaaaag aagaaatcat gaaagcttgt gaaatagctg
    10081 ttgaagctgg attagaattt gttaaaacat caacaggatt ttcaaaatca ggtgcaacat
    10141 ttgaagatgt taaactaatg aagtcagttg ttaaagacaa tgctttagtt aaagcagctg
    10201 gtggagttag aacatttgaa gatgctcaaa aaatgattga agcaggagct gaccgcttag
    10261 gaacaagtgg tggagtagct attattaaag gtgaagaaaa caacgcgagt tactaaaact
    10321 agcgtttttt tattttgctc atttttatta aaagtttgca aaaaggaaca taaaaattct
    10381 aattattgat actaaagtta ttaaaaagaa gattttggtt gattttataa aggtcataga
    10441 atataatatt ttagcatgtg tattttgtgt gctcatttac aaccgtctct gcattggccg
    10501 ggaatcgaac ccgggcctcc cgcgtggcag gcgagaattc taccactgaa ccaccaatgc
    10561 cggtgtttcg tcctttccac aagatatata aagccaagaa atcgaaatac tttcaagtta
    10621 cggtaagcat atgatagtcc attttaaaac ataattttaa aactgcaaac tacccaagaa
    10681 attattactt tctacgtcac gtattttgta ctaatatctt tgtgtttaca gtcaaattaa
    10741 ttctaattat ctctctaaca gccttgtatc gtatatgcaa atatgaagga atcatgggaa
    10801 ataggccctc aagcttggcg taactagatc ttgagacact gctttttgct tgtactgggt
    10861 ctctctggtt agaccagatc tgagcctggg agctctctgg ctaactaggg aacccactgc
    10921 ttaagcctca ataaagcttg ccttgagtgc ttcaagtagt gtgtgcccgt ctgttgtgtg
    10981 actctggtaa ctagagatcc ctcagaccct tttagtcagt gtggaaaatc tctagcagta
    11041 cgtatagtag ttcatgtcat cttattattc agtatttata acttgcaaag aaatgaatat
    11101 cagagagtga gaggaacttg tttattgcag cttataatgg ttacaaataa agcaatagca
    11161 tcacaaattt cacaaataaa gcattttttt cactgcattc tagttgtggt ttgtccaaac
    11221 tcatcaatgt atcttatcat gtctggctct agctatcccg cccctaactc cgcccatccc
    11281 gcccctaact ccgcccagtt ccgcccattc tccgccccat ggctgactaa ttttttttat
    11341 ttatgcagag gccgaggccg cctcggcctc tgagctattc cagaagtagt gaggaggctt
    11401 ttttggaggc ctagggacgt acccaattcg ccctatagtg agtcgtatta cgcgcgctca
    11461 ctggccgtcg ttttacaacg tcgtgactgg gaaaaccctg gcgttaccca acttaatcgc
    11521 cttgcagcac atcccccttt cgccagctgg cgtaatagcg aagaggcccg caccgatcgc
    11581 ccttcccaac agttgcgcag cctgaatggc gaatgggacg cgccctgtag cggcgcatta
    11641 agcgcggcgg gtgtggtggt tacgcgcagc gtgaccgcta cacttgccag cgccctagcg
    11701 cccgctcctt tcgctttctt cccttccttt ctcgccacgt tcgccggctt tccccgtcaa
    11761 gctctaaatc gggggctccc tttagggttc cgatttagtg ctttacggca cctcgacccc
    11821 aaaaaacttg attagggtga tggttcacgt agtgggccat cgccctgata gacggttttt
    11881 cgccctttga cgttggagtc cacgttcttt aatagtggac tcttgttcca aactggaaca
    11941 acactcaacc ctatctcggt ctattctttt gatttataag ggattttgcc gatttcggcc
    12001 tattggttaa aaaatgagct gatttaacaa aaatttaacg cgaattttaa caaaatatta
    12061 acgcttacaa tttaggtggc acttttcggg gaaatgtgcg cggaacccct atttgtttat
    12121 ttttctaaat acattcaaat atgtatccgc tcatgagaca ataaccctga taaatgcttc
    12181 aataatattg aaaaaggaag agtatgagta ttcaacattt ccgtgtcgcc cttattccct
    12241 tttttgcggc attttgcctt cctgtttttg ctcacccaga aacgctggtg aaagtaaaag
    12301 atgctgaaga tcagttgggt gcacgagtgg gttacatcga actggatctc aacagcggta
    12361 agatccttga gagttttcgc cccgaagaac gttttccaat gatgagcact tttaaagttc
    12421 tgctatgtgg cgcggtatta tcccgtattg acgccgggca agagcaactc ggtcgccgca
    12481 tacactattc tcagaatgac ttggttgagt actcaccagt cacagaaaag catcttacgg
    12541 atggcatgac agtaagagaa ttatgcagtg ctgccataac catgagtgat aacactgcgg
    12601 ccaacttact tctgacaacg atcggaggac cgaaggagct aaccgctttt ttgcacaaca
    12661 tgggggatca tgtaactcgc cttgatcgtt gggaaccgga gctgaatgaa gccataccaa
    12721 acgacgagcg tgacaccacg atgcctgtag caatggcaac aacgttgcgc aaactattaa
    12781 ctggcgaact acttactcta gcttcccggc aacaattaat agactggatg gaggcggata
    12841 aagttgcagg accacttctg cgctcggccc ttccggctgg ctggtttatt gctgataaat
    12901 ctggagccgg tgagcgtggg tctcgcggta tcattgcagc actggggcca gatggtaagc
    12961 cctcccgtat cgtagttatc tacacgacgg ggagtcaggc aactatggat gaacgaaata
    13021 gacagatcgc tgagataggt gcctcactga ttaagcattg gtaactgtca gaccaagttt
    13081 actcatatat actttagatt gatttaaaac ttcattttta atttaaaagg atctaggtga
    13141 agatcctttt tgataatctc atgaccaaaa tcccttaacg tgagttttcg ttccactgag
    13201 cgtcagaccc cgtagaaaag atcaaaggat cttcttgaga tccttttttt ctgcgcgtaa
    13261 tctgctgctt gcaaacaaaa aaaccaccgc taccagcggt ggtttgtttg ccggatcaag
    13321 agctaccaac tctttttccg aaggtaactg gcttcagcag agcgcagata ccaaatactg
    13381 ttcttctagt gtagccgtag ttaggccacc acttcaagaa ctctgtagca ccgcctacat
    13441 acctcgctct gctaatcctg ttaccagtgg ctgctgccag tggcgataag tcgtgtctta
    13501 ccgggttgga ctcaagacga tagttaccgg ataaggcgca gcggtcgggc tgaacggggg
    13561 gttcgtgcac acagcccagc ttggagcgaa cgacctacac cgaactgaga tacctacagc
    13621 gtgagctatg agaaagcgcc acgcttcccg aagggagaaa ggcggacagg tatccggtaa
    13681 gcggcagggt cggaacagga gagcgcacga gggagcttcc agggggaaac gcctggtatc
    13741 tttatagtcc tgtcgggttt cgccacctct gacttgagcg tcgatttttg tgatgctcgt
    13801 caggggggcg gagcctatgg aaaaacgcca gcaacgcggc ctttttacgg ttcctggcct
    13861 tttgctggcc ttttgctcac atgttctttc ctgcgttatc ccctgattct gtggataacc
    13921 gtattaccgc ctttgagtga gctgataccg ctcgccgcag ccgaacgacc gagcgcagcg
    13981 agtcagtgag cgaggaagcg gaagagcgcc caatacgcaa accgcctctc cccgcgcgtt
    14041 ggccgattca ttaatgcagc tggcacgaca ggtttcccga ctggaaagcg ggcagtgagc
    14101 gcaacgcaat taatgtgagt tagctcactc attaggcacc ccaggcttta cactttatgc
    14161 ttccggctcg tatgttgtgt ggaattgtga gcggataaca atttcacaca ggaaacagct
    14221 atgaccatga ttacgccaag cgcgcaatta accctcacta aagggaacaa aagctggagc
    14281 tgcaagctta agacattgat tattgactag ttattaatag taatcaatta cggggtcatt
    14341 agttcatagc ccatatatgg agttccgcgt tacataactt acggtaaatg gcccgcctgg
    14401 ctgaccgccc aacgaccccc gcccattgac gtcaataatg acgtatgttc ccatagtaac
    14461 gccaataggg actttccatt gacgtcaatg ggtggagtat ttacggtaaa ctgcccactt
    14521 ggcagtacat caagtgtatc atatgccaag tacgccccct attgacgtca atgacggtaa
    14581 atggcccgcc tggcattatg cccagtacat gaccttatgg gactttccta cttggcagta
    14641 catctacgta ttagtcatcg ctattaccat ggtgatgcgg ttttggcagt acatcaatgg
    14701 gcgtggatag cggtttgact cacggggatt tccaagtctc caccccattg acgtcaatgg
    14761 gagtttgttt tggcaccaaa atcaacggga ctttccaaaa tgtcgtaaca actccgcccc
    14821 attgacgcaa atgggcggta ggcgtgtacg gtgggaggtc tatataagca gcgcgttttg
    14881 cctgtact
//
