LOCUS       sequence-361387-       15273 bp    DNA     circular SYN 15-DEC-2025
DEFINITION  Expresses Cas9 and guide RNA, contains intact LTR.
ACCESSION   .
VERSION     .
KEYWORDS    .
SOURCE      synthetic DNA construct
  ORGANISM  synthetic DNA construct
REFERENCE   1  (bases 1 to 15273)
  AUTHORS   Iaffaldano BJ, Marino MP, Reiser J
  TITLE     CRISPR library screening to develop HEK293-derived cell lines with 
            improved lentiviral vector titers.
  JOURNAL   Front Genome Ed. 2023 Jul 13;5:1218328. doi: 
            10.3389/fgeed.2023.1218328. eCollection 2023.
  PUBMED    37520398
REFERENCE   2  (bases 1 to 15273)
  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: "Front 
            Genome Ed."; date: "2023-07-13"; volume: "5:1218328. doi"; pages: " 
            10.3389/fgeed.2023.1218328. eCollection 2023"
COMMENT     Sequence Label: lenticrispr-wt-ltr-puro
FEATURES             Location/Qualifiers
     source          1..15273
                     /mol_type="other DNA"
                     /organism="synthetic DNA construct"
     CDS             complement(join(223..1014,1015..1083))
                     /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     846..865
                     /label=Amp-R
                     /note="Ampicillin resistance gene, reverse primer"
     promoter        complement(1084..1188)
                     /gene="bla"
                     /label=AmpR promoter
     primer_bind     complement(1263..1282)
                     /label=pRS-marker
                     /note="pRS vectors, use to sequence yeast selectable 
                     marker"
     enhancer        1454..1833
                     /label=CMV enhancer
                     /note="human cytomegalovirus immediate early enhancer"
     promoter        1835..2033
                     /label=CMV promoter
                     /note="human cytomegalovirus (CMV) immediate early 
                     promoter"
     primer_bind     1988..2008
                     /label=CMV-F
                     /note="Human CMV immediate early promoter, forward primer"
     LTR             2051..2231
                     /label=5' LTR (truncated)
                     /note="truncated 5' long terminal repeat (LTR) from HIV-1"
     misc_feature    2278..2403
                     /label=HIV-1 Psi
                     /note="packaging signal of human immunodeficiency virus 
                     type 1"
     misc_feature    2896..3129
                     /label=RRE
                     /note="The Rev response element (RRE) of HIV-1 allows for 
                     Rev-dependent mRNA export from the nucleus to the 
                     cytoplasm."
     CDS             3314..3358
                     /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    3656..3773
                     /label=cPPT/CTS
                     /note="central polypurine tract and central termination 
                     sequence of HIV-1"
     promoter        3824..4072
                     /label=U6 promoter
                     /note="RNA polymerase III promoter for human U6 snRNA 
                     (Domitrovich & Kunkel, 2003)"
     primer_bind     3824..3844
                     /label=hU6-F
                     /note="Human U6 promoter, forward primer"
     primer_bind     3995..4014
                     /label=LKO.1 5'
                     /note="Human U6 promoter, forward primer"
     misc_RNA        5954..6029
                     /label=gRNA scaffold
                     /note="guide RNA scaffold for the Streptococcus pyogenes 
                     CRISPR/Cas9 system"
     promoter        6091..6302
                     /label=EF-1-alpha core promoter
                     /note="core promoter for human elongation factor 
                     EF-1-alpha"
     regulatory      6321..6330
                     /label=Kozak sequence
                     /note="vertebrate consensus sequence for strong initiation 
                     of translation (Kozak, 1987)"
                     /regulatory_class="other"
     CDS             6327..10430
                     /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             6327..10430
                     /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             10431..10478
                     /codon_start=1
                     /product="bipartite nuclear localization signal from 
                     nucleoplasmin"
                     /label=nucleoplasmin NLS
                     /translation="KRPAATKKAGQAKKKK"
     CDS             10479..10502
                     /codon_start=1
                     /product="FLAG(R) epitope tag, followed by an enterokinase 
                     cleavage site"
                     /label=FLAG
                     /translation="DYKDDDDK"
     CDS             10512..10568
                     /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             10569..11165
                     /codon_start=1
                     /gene="pac from Streptomyces alboniger"
                     /product="puromycin N-acetyltransferase"
                     /label=PuroR
                     /note="confers resistance to puromycin"
                     /translation="TEYKPTVRLATRDDVPRAVRTLAAAFADYPATRHTVDPDRHIERV
                     TELQELFLTRVGLDIGKVWVADDGAAVAVWTTPESVEAGAVFAEIGPRMAELSGSRLAA
                     QQQMEGLLAPHRPKEPAWFLATVGVSPDHQGKGLGSAVVLPGVEAAERAGVPAFLETSA
                     PRNLPFYERLGFTVTADVEVPEGPRTWCMTRKPGA"
     primer_bind     11062..11082
                     /label=Puro-F
                     /note="Puromycin resistance gene, forward primer"
     misc_feature    11181..11769
                     /label=WPRE
                     /note="woodchuck hepatitis virus posttranscriptional 
                     regulatory element"
     primer_bind     complement(11234..11254)
                     /label=WPRE-R
                     /note="WPRE, reverse primer"
     CDS             complement(11652..11663)
                     /codon_start=1
                     /product="Factor Xa recognition and cleavage site"
                     /label=Factor Xa site
                     /translation="IEGR"
     LTR             11841..12474
                     /label=5' LTR
                     /note="5' long terminal repeat (LTR) from HIV-1"
     primer_bind     complement(12500..12517)
                     /label=BGH-rev
                     /note="Bovine growth hormone terminator, reverse primer. 
                     Also called BGH reverse"
     polyA_signal    12506..12730
                     /label=bGH poly(A) signal
                     /note="bovine growth hormone polyadenylation signal"
     rep_origin      12776..13204
                     /direction=RIGHT
                     /label=f1 ori
                     /note="f1 bacteriophage origin of replication; arrow 
                     indicates direction of (+) strand synthesis"
     primer_bind     complement(12863..12882)
                     /label=F1ori-R
                     /note="F1 origin, reverse primer"
     primer_bind     13073..13094
                     /label=F1ori-F
                     /note="F1 origin, forward primer"
     primer_bind     complement(13213..13233)
                     /label=pBABE 3'
                     /note="SV40 enhancer, reverse primer for pBABE vectors"
     promoter        13218..13547
                     /label=SV40 promoter
                     /note="SV40 enhancer and early promoter"
     rep_origin      13398..13533
                     /label=SV40 ori
                     /note="SV40 origin of replication"
     primer_bind     13460..13479
                     /label=SV40pro-F
                     /note="SV40 promoter/origin, forward primer"
     promoter        13595..13642
                     /label=EM7 promoter
                     /note="synthetic bacterial promoter "
     CDS             13661..14035
                     /codon_start=1
                     /gene="Sh ble from Streptoalloteichus hindustanus"
                     /product="antibiotic-binding protein"
                     /label=BleoR
                     /note="confers resistance to bleomycin, phleomycin, and 
                     Zeocin(TM)"
                     /translation="MAKLTSAVPVLTARDVAGAVEFWTDRLGFSRDFVEDDFAGVVRDD
                     VTLFISAVQDQVVPDNTLAWVWVRGLDELYAEWSEVVSTNFRDASGPAMTEIGEQPWGR
                     EFALRDPAGNCVHFVAEEQD"
     polyA_signal    14165..14286
                     /label=SV40 poly(A) signal
                     /note="SV40 polyadenylation signal"
     primer_bind     complement(14202..14221)
                     /label=SV40pA-R
                     /note="SV40 polyA, reverse primer"
     primer_bind     14256..14275
                     /label=EBV-rev
                     /note="SV40 polyA terminator, reverse primer"
     primer_bind     complement(14335..14351)
                     /label=M13 rev
                     /note="common sequencing primer, one of multiple similar 
                     variants"
     primer_bind     complement(14335..14351)
                     /label=M13 Reverse
                     /note="In lacZ gene. Also called M13-rev"
     primer_bind     complement(14348..14370)
                     /label=M13/pUC Reverse
                     /note="In lacZ gene"
     protein_bind    14359..14375
                     /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)."
     promoter        complement(join(14383..14389,14390..14407,14408..14413))
                     /label=lac promoter
                     /note="promoter for the E. coli lac operon"
     protein_bind    14428..14449
                     /label=CAP binding site
                     /bound_moiety="E. coli catabolite activator protein"
                     /note="CAP binding activates transcription in the presence 
                     of cAMP."
     primer_bind     complement(14566..14583)
                     /label=L4440
                     /note="L4440 vector, forward primer"
     rep_origin      complement(join(14737..15273,1..52))
                     /direction=LEFT
                     /label=ori
                     /note="high-copy-number ColE1/pMB1/pBR322/pUC origin of 
                     replication"
     primer_bind     complement(14817..14836)
                     /label=pBR322ori-F
                     /note="pBR322 origin, forward primer"
ORIGIN
        1 tggttttttt gtttgcaagc agcagattac gcgcagaaaa aaaggatctc aagaagatcc
       61 tttgatcttt tctacggggt ctgacgctca gtggaacgaa aactcacgtt aagggatttt
      121 ggtcatgaga ttatcaaaaa ggatcttcac ctagatcctt ttaaattaaa aatgaagttt
      181 taaatcaatc taaagtatat atgagtaaac ttggtctgac agttaccaat gcttaatcag
      241 tgaggcacct atctcagcga tctgtctatt tcgttcatcc atagttgcct gactccccgt
      301 cgtgtagata actacgatac gggagggctt accatctggc cccagtgctg caatgatacc
      361 gcgagaccca cgctcaccgg ctccagattt atcagcaata aaccagccag ccggaagggc
      421 cgagcgcaga agtggtcctg caactttatc cgcctccatc cagtctatta attgttgccg
      481 ggaagctaga gtaagtagtt cgccagttaa tagtttgcgc aacgttgttg ccattgctac
      541 aggcatcgtg gtgtcacgct cgtcgtttgg tatggcttca ttcagctccg gttcccaacg
      601 atcaaggcga gttacatgat cccccatgtt gtgcaaaaaa gcggttagct ccttcggtcc
      661 tccgatcgtt gtcagaagta agttggccgc agtgttatca ctcatggtta tggcagcact
      721 gcataattct cttactgtca tgccatccgt aagatgcttt tctgtgactg gtgagtactc
      781 aaccaagtca ttctgagaat agtgtatgcg gcgaccgagt tgctcttgcc cggcgtcaat
      841 acgggataat accgcgccac atagcagaac tttaaaagtg ctcatcattg gaaaacgttc
      901 ttcggggcga aaactctcaa ggatcttacc gctgttgaga tccagttcga tgtaacccac
      961 tcgtgcaccc aactgatctt cagcatcttt tactttcacc agcgtttctg ggtgagcaaa
     1021 aacaggaagg caaaatgccg caaaaaaggg aataagggcg acacggaaat gttgaatact
     1081 catactcttc ctttttcaat attattgaag catttatcag ggttattgtc tcatgagcgg
     1141 atacatattt gaatgtattt agaaaaataa acaaataggg gttccgcgca catttccccg
     1201 aaaagtgcca cctgacgtcg acggatcggg agatctcccg atcccctatg gtgcactctc
     1261 agtacaatct gctctgatgc cgcatagtta agccagtatc tgctccctgc ttgtgtgttg
     1321 gaggtcgctg agtagtgcgc gagcaaaatt taagctacaa caaggcaagg cttgaccgac
     1381 aattgcatga agaatctgct tagggttagg cgttttgcgc tgcttcgcga tgtacgggcc
     1441 agatatacgc gttgacattg attattgact agttattaat agtaatcaat tacggggtca
     1501 ttagttcata gcccatatat ggagttccgc gttacataac ttacggtaaa tggcccgcct
     1561 ggctgaccgc ccaacgaccc ccgcccattg acgtcaataa tgacgtatgt tcccatagta
     1621 acgccaatag ggactttcca ttgacgtcaa tgggtggagt atttacggta aactgcccac
     1681 ttggcagtac atcaagtgta tcatatgcca agtacgcccc ctattgacgt caatgacggt
     1741 aaatggcccg cctggcatta tgcccagtac atgaccttat gggactttcc tacttggcag
     1801 tacatctacg tattagtcat cgctattacc atggtgatgc ggttttggca gtacatcaat
     1861 gggcgtggat agcggtttga ctcacgggga tttccaagtc tccaccccat tgacgtcaat
     1921 gggagtttgt tttggcacca aaatcaacgg gactttccaa aatgtcgtaa caactccgcc
     1981 ccattgacgc aaatgggcgg taggcgtgta cggtgggagg tctatataag cagcgcgttt
     2041 tgcctgtact gggtctctct ggttagacca gatctgagcc tgggagctct ctggctaact
     2101 agggaaccca ctgcttaagc ctcaataaag cttgccttga gtgcttcaag tagtgtgtgc
     2161 ccgtctgttg tgtgactctg gtaactagag atccctcaga cccttttagt cagtgtggaa
     2221 aatctctagc agtggcgccc gaacagggac ttgaaagcga aagggaaacc agaggagctc
     2281 tctcgacgca ggactcggct tgctgaagcg cgcacggcaa gaggcgaggg gcggcgactg
     2341 gtgagtacgc caaaaatttt gactagcgga ggctagaagg agagagatgg gtgcgagagc
     2401 gtcagtatta agcgggggag aattagatcg cgatgggaaa aaattcggtt aaggccaggg
     2461 ggaaagaaaa aatataaatt aaaacatata gtatgggcaa gcagggagct agaacgattc
     2521 gcagttaatc ctggcctgtt agaaacatca gaaggctgta gacaaatact gggacagcta
     2581 caaccatccc ttcagacagg atcagaagaa cttagatcat tatataatac agtagcaacc
     2641 ctctattgtg tgcatcaaag gatagagata aaagacacca aggaagcttt agacaagata
     2701 gaggaagagc aaaacaaaag taagaccacc gcacagcaag cggccgctga tcttcagacc
     2761 tggaggagga gatatgaggg acaattggag aagtgaatta tataaatata aagtagtaaa
     2821 aattgaacca ttaggagtag cacccaccaa ggcaaagaga agagtggtgc agagagaaaa
     2881 aagagcagtg ggaataggag ctttgttcct tgggttcttg ggagcagcag gaagcactat
     2941 gggcgcagcg tcaatgacgc tgacggtaca ggccagacaa ttattgtctg gtatagtgca
     3001 gcagcagaac aatttgctga gggctattga ggcgcaacag catctgttgc aactcacagt
     3061 ctggggcatc aagcagctcc aggcaagaat cctggctgtg gaaagatacc taaaggatca
     3121 acagctcctg gggatttggg gttgctctgg aaaactcatt tgcaccactg ctgtgccttg
     3181 gaatgctagt tggagtaata aatctctgga acagatttgg aatcacacga cctggatgga
     3241 gtgggacaga gaaattaaca attacacaag cttaatacac tccttaattg aagaatcgca
     3301 aaaccagcaa gaaaagaatg aacaagaatt attggaatta gataaatggg caagtttgtg
     3361 gaattggttt aacataacaa attggctgtg gtatataaaa ttattcataa tgatagtagg
     3421 aggcttggta ggtttaagaa tagtttttgc tgtactttct atagtgaata gagttaggca
     3481 gggatattca ccattatcgt ttcagaccca cctcccaacc ccgaggggac ccgacaggcc
     3541 cgaaggaata gaagaagaag gtggagagag agacagagac agatccattc gattagtgaa
     3601 cggatcggca ctgcgtgcgc caattctgca gacaaatggc agtattcatc cacaatttta
     3661 aaagaaaagg ggggattggg gggtacagtg caggggaaag aatagtagac ataatagcaa
     3721 cagacataca aactaaagaa ttacaaaaac aaattacaaa aattcaaaat tttcgggttt
     3781 attacaggga cagcagagat ccagtttggt taattaaggt accgagggcc tatttcccat
     3841 gattccttca tatttgcata tacgatacaa ggctgttaga gagataatta gaattaattt
     3901 gactgtaaac acaaagatat tagtacaaaa tacgtgacgt agaaagtaat aatttcttgg
     3961 gtagtttgca gttttaaaat tatgttttaa aatggactat catatgctta ccgtaacttg
     4021 aaagtatttc gatttcttgg ctttatatat cttgtggaaa ggacgaaaca ccggagacgg
     4081 ttgtaaatga gcacacaaaa tacacatgct aaaatattat attctatgac ctttataaaa
     4141 tcaaccaaaa tcttcttttt aataacttta gtatcaataa ttagaatttt tatgttcctt
     4201 tttgcaaact tttaataaaa atgagcaaaa taaaaaaacg ctagttttag taactcgcgt
     4261 tgttttcttc acctttaata atagctactc caccacttgt tcctaagcgg tcagctcctg
     4321 cttcaatcat tttttgagca tcttcaaatg ttctaactcc accagctgct ttaactaaag
     4381 cattgtcttt aacaactgac ttcattagtt taacatcttc aaatgttgca cctgattttg
     4441 aaaatcctgt tgatgtttta acaaattcta atccagcttc aacagctatt tcacaagctt
     4501 tcatgatttc ttcttttgtt aataaacaat tttccataat acatttaaca acatgtgatc
     4561 cagctgcttt ttttacagct ttcatgtctt ctaaaactaa ttcataattt ttgtctttta
     4621 atgcaccaat atttaatacc atatcaattt ctgttgcacc atctttaatt gcttcagaaa
     4681 cttcgaatgc ttttgtagct gttgtgcatg cacctagagg aaaacctaca acatttgtta
     4741 ttcctacatt tgtgcctttt aataattctt tacaatagct tgttcaatat gaattaacac
     4801 aaactgttgc aaaatcaaat tcaattgctt catcacataa ttgtttaatt tcagctttcg
     4861 tagcatcttg ttttaataat gtgtgatcta tatatttgtt tagtttcatt ttttctccta
     4921 tatattcatt tttaatttta attctttaat aatttcgtct actttaactt tagcgttttg
     4981 aacagattca ccaacaccta taaaataaat ttttagttta ggttcagttc cacttgggcg
     5041 aacagcaaat catgacttat cttctaaata aaattttagt aagtcttgtc ctggcatatt
     5101 atacattcca tcgatgtagt cttcaacatt aacaacttta agtccagcaa tttgagttaa
     5161 gggtgttgct ctcaatgatt tcattaatgg ttcaattttt aatttctttt cttctggttt
     5221 aaaattcaag tttaaagtga aagtgtaata tgcacccatt tctttaaata aatcttctaa
     5281 atagtctact aatgttttat tttgtttttt ataaaatcaa gcagcctctg ctattaatat
     5341 agaagcttgt attccatctt tatctctagc tgagtcatca attacatatc cataactttc
     5401 ttcataagca aaaacaaaat ttaatccgtt atcttcttct ttagcaattt ctctacccat
     5461 tcatttaaat ccagttaaag tttttacaat attaactcca tatttttcat gagcgattct
     5521 atcacccaaa tcacttgtta caaaacttga atatagagcc ggattttttg gaatgctatt
     5581 taagcgtttt agatttgata attttcaatc aattaaaatt ggtcctgttt gatttccatc
     5641 taatcttaca aaatgaccat catgttttat tgccattcca aatctgtcag catctgggtc
     5701 attcataata ataatatctg catcatgttt aataccatat tcaagcggta tttttcatgc
     5761 aggatcaaat tctggatttg gatttacaac atttttaaat gtttcatctt caaatgcatg
     5821 ctcttcaacc tcaataacgt tatatcctga ttcacgtaat atttttgggg taaatttagt
     5881 tcctgttcca ttaactgcgc taaaaataat ttttaaatct tttttagctt cttgctcttt
     5941 tttgtacgtc tctgttttag agctagaaat agcaagttaa aataaggcta gtccgttatc
     6001 aacttgaaaa agtggcaccg agtcggtgct tttttgaatt cgctagctag gtcttgaaag
     6061 gagtgggaat tggctccggt gcccgtcagt gggcagagcg cacatcgccc acagtccccg
     6121 agaagttggg gggaggggtc ggcaattgat ccggtgccta gagaaggtgg cgcggggtaa
     6181 actgggaaag tgatgtcgtg tactggctcc gcctttttcc cgagggtggg ggagaaccgt
     6241 atataagtgc agtagtcgcc gtgaacgttc tttttcgcaa cgggtttgcc gccagaacac
     6301 aggaccggtt ctagagcgct gccaccatgg acaagaagta cagcatcggc ctggacatcg
     6361 gcaccaactc tgtgggctgg gccgtgatca ccgacgagta caaggtgccc agcaagaaat
     6421 tcaaggtgct gggcaacacc gaccggcaca gcatcaagaa gaacctgatc ggagccctgc
     6481 tgttcgacag cggcgaaaca gccgaggcca cccggctgaa gagaaccgcc agaagaagat
     6541 acaccagacg gaagaaccgg atctgctatc tgcaagagat cttcagcaac gagatggcca
     6601 aggtggacga cagcttcttc cacagactgg aagagtcctt cctggtggaa gaggataaga
     6661 agcacgagcg gcaccccatc ttcggcaaca tcgtggacga ggtggcctac cacgagaagt
     6721 accccaccat ctaccacctg agaaagaaac tggtggacag caccgacaag gccgacctgc
     6781 ggctgatcta tctggccctg gcccacatga tcaagttccg gggccacttc ctgatcgagg
     6841 gcgacctgaa ccccgacaac agcgacgtgg acaagctgtt catccagctg gtgcagacct
     6901 acaaccagct gttcgaggaa aaccccatca acgccagcgg cgtggacgcc aaggccatcc
     6961 tgtctgccag actgagcaag agcagacggc tggaaaatct gatcgcccag ctgcccggcg
     7021 agaagaagaa tggcctgttc ggaaacctga ttgccctgag cctgggcctg acccccaact
     7081 tcaagagcaa cttcgacctg gccgaggatg ccaaactgca gctgagcaag gacacctacg
     7141 acgacgacct ggacaacctg ctggcccaga tcggcgacca gtacgccgac ctgtttctgg
     7201 ccgccaagaa cctgtccgac gccatcctgc tgagcgacat cctgagagtg aacaccgaga
     7261 tcaccaaggc ccccctgagc gcctctatga tcaagagata cgacgagcac caccaggacc
     7321 tgaccctgct gaaagctctc gtgcggcagc agctgcctga gaagtacaaa gagattttct
     7381 tcgaccagag caagaacggc tacgccggct acattgacgg cggagccagc caggaagagt
     7441 tctacaagtt catcaagccc atcctggaaa agatggacgg caccgaggaa ctgctcgtga
     7501 agctgaacag agaggacctg ctgcggaagc agcggacctt cgacaacggc agcatccccc
     7561 accagatcca cctgggagag ctgcacgcca ttctgcggcg gcaggaagat ttttacccat
     7621 tcctgaagga caaccgggaa aagatcgaga agatcctgac cttccgcatc ccctactacg
     7681 tgggccctct ggccagggga aacagcagat tcgcctggat gaccagaaag agcgaggaaa
     7741 ccatcacccc ctggaacttc gaggaagtgg tggacaaggg cgcttccgcc cagagcttca
     7801 tcgagcggat gaccaacttc gataagaacc tgcccaacga gaaggtgctg cccaagcaca
     7861 gcctgctgta cgagtacttc accgtgtata acgagctgac caaagtgaaa tacgtgaccg
     7921 agggaatgag aaagcccgcc ttcctgagcg gcgagcagaa aaaggccatc gtggacctgc
     7981 tgttcaagac caaccggaaa gtgaccgtga agcagctgaa agaggactac ttcaagaaaa
     8041 tcgagtgctt cgactccgtg gaaatctccg gcgtggaaga tcggttcaac gcctccctgg
     8101 gcacatacca cgatctgctg aaaattatca aggacaagga cttcctggac aatgaggaaa
     8161 acgaggacat tctggaagat atcgtgctga ccctgacact gtttgaggac agagagatga
     8221 tcgaggaacg gctgaaaacc tatgcccacc tgttcgacga caaagtgatg aagcagctga
     8281 agcggcggag atacaccggc tggggcaggc tgagccggaa gctgatcaac ggcatccggg
     8341 acaagcagtc cggcaagaca atcctggatt tcctgaagtc cgacggcttc gccaacagaa
     8401 acttcatgca gctgatccac gacgacagcc tgacctttaa agaggacatc cagaaagccc
     8461 aggtgtccgg ccagggcgat agcctgcacg agcacattgc caatctggcc ggcagccccg
     8521 ccattaagaa gggcatcctg cagacagtga aggtggtgga cgagctcgtg aaagtgatgg
     8581 gccggcacaa gcccgagaac atcgtgatcg aaatggccag agagaaccag accacccaga
     8641 agggacagaa gaacagccgc gagagaatga agcggatcga agagggcatc aaagagctgg
     8701 gcagccagat cctgaaagaa caccccgtgg aaaacaccca gctgcagaac gagaagctgt
     8761 acctgtacta cctgcagaat gggcgggata tgtacgtgga ccaggaactg gacatcaacc
     8821 ggctgtccga ctacgatgtg gaccatatcg tgcctcagag ctttctgaag gacgactcca
     8881 tcgacaacaa ggtgctgacc agaagcgaca agaaccgggg caagagcgac aacgtgccct
     8941 ccgaagaggt cgtgaagaag atgaagaact actggcggca gctgctgaac gccaagctga
     9001 ttacccagag aaagttcgac aatctgacca aggccgagag aggcggcctg agcgaactgg
     9061 ataaggccgg cttcatcaag agacagctgg tggaaacccg gcagatcaca aagcacgtgg
     9121 cacagatcct ggactcccgg atgaacacta agtacgacga gaatgacaag ctgatccggg
     9181 aagtgaaagt gatcaccctg aagtccaagc tggtgtccga tttccggaag gatttccagt
     9241 tttacaaagt gcgcgagatc aacaactacc accacgccca cgacgcctac ctgaacgccg
     9301 tcgtgggaac cgccctgatc aaaaagtacc ctaagctgga aagcgagttc gtgtacggcg
     9361 actacaaggt gtacgacgtg cggaagatga tcgccaagag cgagcaggaa atcggcaagg
     9421 ctaccgccaa gtacttcttc tacagcaaca tcatgaactt tttcaagacc gagattaccc
     9481 tggccaacgg cgagatccgg aagcggcctc tgatcgagac aaacggcgaa accggggaga
     9541 tcgtgtggga taagggccgg gattttgcca ccgtgcggaa agtgctgagc atgccccaag
     9601 tgaatatcgt gaaaaagacc gaggtgcaga caggcggctt cagcaaagag tctatcctgc
     9661 ccaagaggaa cagcgataag ctgatcgcca gaaagaagga ctgggaccct aagaagtacg
     9721 gcggcttcga cagccccacc gtggcctatt ctgtgctggt ggtggccaaa gtggaaaagg
     9781 gcaagtccaa gaaactgaag agtgtgaaag agctgctggg gatcaccatc atggaaagaa
     9841 gcagcttcga gaagaatccc atcgactttc tggaagccaa gggctacaaa gaagtgaaaa
     9901 aggacctgat catcaagctg cctaagtact ccctgttcga gctggaaaac ggccggaaga
     9961 gaatgctggc ctctgccggc gaactgcaga agggaaacga actggccctg ccctccaaat
    10021 atgtgaactt cctgtacctg gccagccact atgagaagct gaagggctcc cccgaggata
    10081 atgagcagaa acagctgttt gtggaacagc acaagcacta cctggacgag atcatcgagc
    10141 agatcagcga gttctccaag agagtgatcc tggccgacgc taatctggac aaagtgctgt
    10201 ccgcctacaa caagcaccgg gataagccca tcagagagca ggccgagaat atcatccacc
    10261 tgtttaccct gaccaatctg ggagcccctg ccgccttcaa gtactttgac accaccatcg
    10321 accggaagag gtacaccagc accaaagagg tgctggacgc caccctgatc caccagagca
    10381 tcaccggcct gtacgagaca cggatcgacc tgtctcagct gggaggcgac aagcgacctg
    10441 ccgccacaaa gaaggctgga caggctaaga agaagaaaga ttacaaagac gatgacgata
    10501 agggatccgg cgcaacaaac ttctctctgc tgaaacaagc cggagatgtc gaagagaatc
    10561 ctggaccgac cgagtacaag cccacggtgc gcctcgccac ccgcgacgac gtccccaggg
    10621 ccgtacgcac cctcgccgcc gcgttcgccg actaccccgc cacgcgccac accgtcgatc
    10681 cggaccgcca catcgagcgg gtcaccgagc tgcaagaact cttcctcacg cgcgtcgggc
    10741 tcgacatcgg caaggtgtgg gtcgcggacg acggcgccgc ggtggcggtc tggaccacgc
    10801 cggagagcgt cgaagcgggg gcggtgttcg ccgagatcgg cccgcgcatg gccgagttga
    10861 gcggttcccg gctggccgcg cagcaacaga tggaaggcct cctggcgccg caccggccca
    10921 aggagcccgc gtggttcctg gccaccgtcg gagtctcgcc cgaccaccag ggcaagggtc
    10981 tgggcagcgc cgtcgtgctc cccggagtgg aggcggccga gcgcgccggg gtgcccgcct
    11041 tcctggagac ctccgcgccc cgcaacctcc ccttctacga gcggctcggc ttcaccgtca
    11101 ccgccgacgt cgaggtgccc gaaggaccgc gcacctggtg catgacccgc aagcccggtg
    11161 cctgaacgcg ttaagtcgac aatcaacctc tggattacaa aatttgtgaa agattgactg
    11221 gtattcttaa ctatgttgct ccttttacgc tatgtggata cgctgcttta atgcctttgt
    11281 atcatgctat tgcttcccgt atggctttca ttttctcctc cttgtataaa tcctggttgc
    11341 tgtctcttta tgaggagttg tggcccgttg tcaggcaacg tggcgtggtg tgcactgtgt
    11401 ttgctgacgc aacccccact ggttggggca ttgccaccac ctgtcagctc ctttccggga
    11461 ctttcgcttt ccccctccct attgccacgg cggaactcat cgccgcctgc cttgcccgct
    11521 gctggacagg ggctcggctg ttgggcactg acaattccgt ggtgttgtcg gggaaatcat
    11581 cgtcctttcc ttggctgctc gcctgtgttg ccacctggat tctgcgcggg acgtccttct
    11641 gctacgtccc ttcggccctc aatccagcgg accttccttc ccgcggcctg ctgccggctc
    11701 tgcggcctct tccgcgtctt cgccttcgcc ctcagacgag tcggatctcc ctttgggccg
    11761 cctccccgcg tcgactttaa gaccaatgac ttacaaggca gctgtagatc ttagccactt
    11821 tttaaaagaa aaggggggac tggaagggct aattcactcc caacgaagac aagatatcct
    11881 tgatctgtgg atctaccaca cacaaggcta cttccctgat tggcagaact acacaccagg
    11941 gccagggatc agatatccac tgacctttgg atggtgctac aagctagtac cagttgagca
    12001 agagaaggta gaagaagcca atgaaggaga gaacacccgc ttgttacacc ctgtgagcct
    12061 gcatgggatg gatgacccgg agagagaagt attagagtgg aggtttgaca gccgcctagc
    12121 atttcatcac atggcccgag agctgcatcc ggagtacttc aagaactgct gacatcgagc
    12181 ttgctacaag ggactttccg ctggggactt tccagggagg cgtggcctgg gcgggactgg
    12241 ggagtggcga gccctcagat gctgcatata agcagctgct ttttgcttgt actgggtctc
    12301 tctggttaga ccagatctga gcctgggagc tctctggcta actagggaac ccactgctta
    12361 agcctcaata aagcttgcct tgagtgcttc aagtagtgtg tgcccgtctg ttgtgtgact
    12421 ctggtaacta gagatccctc agaccctttt agtcagtgtg gaaaatctct agcagggccc
    12481 gtttaaaccc gctgatcagc ctcgactgtg ccttctagtt gccagccatc tgttgtttgc
    12541 ccctcccccg tgccttcctt gaccctggaa ggtgccactc ccactgtcct ttcctaataa
    12601 aatgaggaaa ttgcatcgca ttgtctgagt aggtgtcatt ctattctggg gggtggggtg
    12661 gggcaggaca gcaaggggga ggattgggaa gacaatagca ggcatgctgg ggatgcggtg
    12721 ggctctatgg cttctgaggc ggaaagaacc agctggggct ctagggggta tccccacgcg
    12781 ccctgtagcg gcgcattaag cgcggcgggt gtggtggtta cgcgcagcgt gaccgctaca
    12841 cttgccagcg ccctagcgcc cgctcctttc gctttcttcc cttcctttct cgccacgttc
    12901 gccggctttc cccgtcaagc tctaaatcgg gggctccctt tagggttccg atttagtgct
    12961 ttacggcacc tcgaccccaa aaaacttgat tagggtgatg gttcacgtag tgggccatcg
    13021 ccctgataga cggtttttcg ccctttgacg ttggagtcca cgttctttaa tagtggactc
    13081 ttgttccaaa ctggaacaac actcaaccct atctcggtct attcttttga tttataaggg
    13141 attttgccga tttcggccta ttggttaaaa aatgagctga tttaacaaaa atttaacgcg
    13201 aattaattct gtggaatgtg tgtcagttag ggtgtggaaa gtccccaggc tccccagcag
    13261 gcagaagtat gcaaagcatg catctcaatt agtcagcaac caggtgtgga aagtccccag
    13321 gctccccagc aggcagaagt atgcaaagca tgcatctcaa ttagtcagca accatagtcc
    13381 cgcccctaac tccgcccatc ccgcccctaa ctccgcccag ttccgcccat tctccgcccc
    13441 atggctgact aatttttttt atttatgcag aggccgaggc cgcctctgcc tctgagctat
    13501 tccagaagta gtgaggaggc ttttttggag gcctaggctt ttgcaaaaag ctcccgggag
    13561 cttgtatatc cattttcgga tctgatcagc acgtgttgac aattaatcat cggcatagta
    13621 tatcggcata gtataatacg acaaggtgag gaactaaacc atggccaagt tgaccagtgc
    13681 cgttccggtg ctcaccgcgc gcgacgtcgc cggagcggtc gagttctgga ccgaccggct
    13741 cgggttctcc cgggacttcg tggaggacga cttcgccggt gtggtccggg acgacgtgac
    13801 cctgttcatc agcgcggtcc aggaccaggt ggtgccggac aacaccctgg cctgggtgtg
    13861 ggtgcgcggc ctggacgagc tgtacgccga gtggtcggag gtcgtgtcca cgaacttccg
    13921 ggacgcctcc gggccggcca tgaccgagat cggcgagcag ccgtgggggc gggagttcgc
    13981 cctgcgcgac ccggccggca actgcgtgca cttcgtggcc gaggagcagg actgacacgt
    14041 gctacgagat ttcgattcca ccgccgcctt ctatgaaagg ttgggcttcg gaatcgtttt
    14101 ccgggacgcc ggctggatga tcctccagcg cggggatctc atgctggagt tcttcgccca
    14161 ccccaacttg tttattgcag cttataatgg ttacaaataa agcaatagca tcacaaattt
    14221 cacaaataaa gcattttttt cactgcattc tagttgtggt ttgtccaaac tcatcaatgt
    14281 atcttatcat gtctgtatac cgtcgacctc tagctagagc ttggcgtaat catggtcata
    14341 gctgtttcct gtgtgaaatt gttatccgct cacaattcca cacaacatac gagccggaag
    14401 cataaagtgt aaagcctggg gtgcctaatg agtgagctaa ctcacattaa ttgcgttgcg
    14461 ctcactgccc gctttccagt cgggaaacct gtcgtgccag ctgcattaat gaatcggcca
    14521 acgcgcgggg agaggcggtt tgcgtattgg gcgctcttcc gcttcctcgc tcactgactc
    14581 gctgcgctcg gtcgttcggc tgcggcgagc ggtatcagct cactcaaagg cggtaatacg
    14641 gttatccaca gaatcagggg ataacgcagg aaagaacatg tgagcaaaag gccagcaaaa
    14701 ggccaggaac cgtaaaaagg ccgcgttgct ggcgtttttc cataggctcc gcccccctga
    14761 cgagcatcac aaaaatcgac gctcaagtca gaggtggcga aacccgacag gactataaag
    14821 ataccaggcg tttccccctg gaagctccct cgtgcgctct cctgttccga ccctgccgct
    14881 taccggatac ctgtccgcct ttctcccttc gggaagcgtg gcgctttctc atagctcacg
    14941 ctgtaggtat ctcagttcgg tgtaggtcgt tcgctccaag ctgggctgtg tgcacgaacc
    15001 ccccgttcag cccgaccgct gcgccttatc cggtaactat cgtcttgagt ccaacccggt
    15061 aagacacgac ttatcgccac tggcagcagc cactggtaac aggattagca gagcgaggta
    15121 tgtaggcggt gctacagagt tcttgaagtg gtggcctaac tacggctaca ctagaagaac
    15181 agtatttggt atctgcgctc tgctgaagcc agttaccttc ggaaaaagag ttggtagctc
    15241 ttgatccggc aaacaaacca ccgctggtag cgg
//
