LOCUS       sequence-408735-       13332 bp    DNA     circular SYN 15-DEC-2025
DEFINITION  Expression of a CRISPRi guide targeting BIRC6.
ACCESSION   .
VERSION     .
KEYWORDS    .
SOURCE      synthetic DNA construct
  ORGANISM  synthetic DNA construct
REFERENCE   1  (bases 1 to 13332)
  AUTHORS   Cervia LD, Shibue T, Borah AA, Gaeta B, He L, Leung L, Li N, Moyer 
            SM, Shim BH, Dumont N, Gonzalez A, Bick NR, Kazachkova M, Dempster 
            JM, Krill-Burger JM, Piccioni F, Udeshi ND, Olive ME, Carr SA, Root 
            DE, McFarland JM, Vazquez F, Hahn WC
  TITLE     A Ubiquitination Cascade Regulating the Integrated Stress Response 
            and Survival in Carcinomas.
  JOURNAL   Cancer Discov. 2023 Mar 1;13(3):766-795. doi: 
            10.1158/2159-8290.CD-22-1230.
  PUBMED    36576405
REFERENCE   2  (bases 1 to 13332)
  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"; doi: "10.1158/2159-8290";
            journalName: "Cancer Discov"; date: "2023-03-1- 1"; volume: "13"; 
            issue: "3"; pages: "766-795"
COMMENT     Sequence Label: pXPR_023d-sgCiBIRC6-1
FEATURES             Location/Qualifiers
     source          1..13332
                     /mol_type="other DNA"
                     /organism="synthetic DNA construct"
     enhancer        134..513
                     /label=CMV enhancer
                     /note="human cytomegalovirus immediate early enhancer"
     promoter        515..713
                     /label=CMV promoter
                     /note="human cytomegalovirus (CMV) immediate early 
                     promoter"
     primer_bind     668..688
                     /label=CMV-F
                     /note="Human CMV immediate early promoter, forward primer"
     LTR             731..911
                     /label=5' LTR (truncated)
                     /note="truncated 5' long terminal repeat (LTR) from HIV-1"
     misc_feature    958..1083
                     /label=HIV-1 Psi
                     /note="packaging signal of human immunodeficiency virus 
                     type 1"
     misc_feature    1576..1809
                     /label=RRE
                     /note="The Rev response element (RRE) of HIV-1 allows for 
                     Rev-dependent mRNA export from the nucleus to the 
                     cytoplasm."
     CDS             1994..2038
                     /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    2336..2453
                     /label=cPPT/CTS
                     /note="central polypurine tract and central termination 
                     sequence of HIV-1"
     promoter        2504..2752
                     /label=U6 promoter
                     /note="RNA polymerase III promoter for human U6 snRNA 
                     (Domitrovich & Kunkel, 2003)"
     primer_bind     2504..2524
                     /label=hU6-F
                     /note="Human U6 promoter, forward primer"
     primer_bind     2675..2694
                     /label=LKO.1 5'
                     /note="Human U6 promoter, forward primer"
     misc_RNA        2774..2849
                     /label=gRNA scaffold
                     /note="guide RNA scaffold for the Streptococcus pyogenes 
                     CRISPR/Cas9 system"
     promoter        2911..3122
                     /label=EF-1-alpha core promoter
                     /note="core promoter for human elongation factor 
                     EF-1-alpha"
     protein_bind    3148..3172
                     /gene="mutant version of attB"
                     /label=attB1
                     /bound_moiety="BP Clonase(TM)"
                     /note="recombination site for the Gateway(R) BP reaction"
     CDS             3202..3387
                     /codon_start=1
                     /product="Kruppel-associated box (KRAB) transcriptional 
                     repression domain from the human zinc finger protein ZNF10 
                     (Margolin et al., 1994)"
                     /label=KRAB
                     /translation="RTLVTFKDVFVDFTREEWKLLDTAQQIVYRNVMLENYKNLVSLGY
                     QLTKPDVILRLEKGEEP"
     CDS             join(3409..3435,3436..3438,3439..5925,5926..5928,
                     5929..7512)
                     /codon_start=1
                     /product="catalytically dead mutant of the Cas9 
                     endonuclease from the Streptococcus pyogenes Type II 
                     CRISPR/Cas system"
                     /label=dCas9
                     /note="RNA-guided DNA-binding protein that lacks 
                     endonuclease activity due to the D10A mutation in the RuvC 
                     catalytic domain and the H840A mutation in the HNH 
                     catalytic domain"
                     /translation="MDKKYSIGLAIGTNSVGWAVITDEYKVPSKKFKVLGNTDRHSIKK
                     NLIGALLFDSGETAEATRLKRTARRRYTRRKNRICYLQEIFSNEMAKVDDSFFHRLEES
                     FLVEEDKKHERHPIFGNIVDEVAYHEKYPTIYHLRKKLVDSTDKADLRLIYLALAHMIK
                     FRGHFLIEGDLNPDNSDVDKLFIQLVQTYNQLFEENPINASGVDAKAILSARLSKSRRL
                     ENLIAQLPGEKKNGLFGNLIALSLGLTPNFKSNFDLAEDAKLQLSKDTYDDDLDNLLAQ
                     IGDQYADLFLAAKNLSDAILLSDILRVNTEITKAPLSASMIKRYDEHHQDLTLLKALVR
                     QQLPEKYKEIFFDQSKNGYAGYIDGGASQEEFYKFIKPILEKMDGTEELLVKLNREDLL
                     RKQRTFDNGSIPHQIHLGELHAILRRQEDFYPFLKDNREKIEKILTFRIPYYVGPLARG
                     NSRFAWMTRKSEETITPWNFEEVVDKGASAQSFIERMTNFDKNLPNEKVLPKHSLLYEY
                     FTVYNELTKVKYVTEGMRKPAFLSGEQKKAIVDLLFKTNRKVTVKQLKEDYFKKIECFD
                     SVEISGVEDRFNASLGTYHDLLKIIKDKDFLDNEENEDILEDIVLTLTLFEDREMIEER
                     LKTYAHLFDDKVMKQLKRRRYTGWGRLSRKLINGIRDKQSGKTILDFLKSDGFANRNFM
                     QLIHDDSLTFKEDIQKAQVSGQGDSLHEHIANLAGSPAIKKGILQTVKVVDELVKVMGR
                     HKPENIVIEMARENQTTQKGQKNSRERMKRIEEGIKELGSQILKEHPVENTQLQNEKLY
                     LYYLQNGRDMYVDQELDINRLSDYDVDAIVPQSFLKDDSIDNKVLTRSDKNRGKSDNVP
                     SEEVVKKMKNYWRQLLNAKLITQRKFDNLTKAERGGLSELDKAGFIKRQLVETRQITKH
                     VAQILDSRMNTKYDENDKLIREVKVITLKSKLVSDFRKDFQFYKVREINNYHHAHDAYL
                     NAVVGTALIKKYPKLESEFVYGDYKVYDVRKMIAKSEQEIGKATAKYFFYSNIMNFFKT
                     EITLANGEIRKRPLIETNGETGEIVWDKGRDFATVRKVLSMPQVNIVKKTEVQTGGFSK
                     ESILPKRNSDKLIARKKDWDPKKYGGFDSPTVAYSVLVVAKVEKGKSKKLKSVKELLGI
                     TIMERSSFEKNPIDFLEAKGYKEVKKDLIIKLPKYSLFELENGRKRMLASAGELQKGNE
                     LALPSKYVNFLYLASHYEKLKGSPEDNEQKQLFVEQHKHYLDEIIEQISEFSKRVILAD
                     ANLDKVLSAYNKHRDKPIREQAENIIHLFTLTNLGAPAAFKYFDTTIDRKRYTSTKEVL
                     DATLIHQSITGLYETRIDLSQLGGD"
     CDS             3409..7512
                     /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="MDKKYSIGLAIGTNSVGWAVITDEYKVPSKKFKVLGNTDRHSIKK
                     NLIGALLFDSGETAEATRLKRTARRRYTRRKNRICYLQEIFSNEMAKVDDSFFHRLEES
                     FLVEEDKKHERHPIFGNIVDEVAYHEKYPTIYHLRKKLVDSTDKADLRLIYLALAHMIK
                     FRGHFLIEGDLNPDNSDVDKLFIQLVQTYNQLFEENPINASGVDAKAILSARLSKSRRL
                     ENLIAQLPGEKKNGLFGNLIALSLGLTPNFKSNFDLAEDAKLQLSKDTYDDDLDNLLAQ
                     IGDQYADLFLAAKNLSDAILLSDILRVNTEITKAPLSASMIKRYDEHHQDLTLLKALVR
                     QQLPEKYKEIFFDQSKNGYAGYIDGGASQEEFYKFIKPILEKMDGTEELLVKLNREDLL
                     RKQRTFDNGSIPHQIHLGELHAILRRQEDFYPFLKDNREKIEKILTFRIPYYVGPLARG
                     NSRFAWMTRKSEETITPWNFEEVVDKGASAQSFIERMTNFDKNLPNEKVLPKHSLLYEY
                     FTVYNELTKVKYVTEGMRKPAFLSGEQKKAIVDLLFKTNRKVTVKQLKEDYFKKIECFD
                     SVEISGVEDRFNASLGTYHDLLKIIKDKDFLDNEENEDILEDIVLTLTLFEDREMIEER
                     LKTYAHLFDDKVMKQLKRRRYTGWGRLSRKLINGIRDKQSGKTILDFLKSDGFANRNFM
                     QLIHDDSLTFKEDIQKAQVSGQGDSLHEHIANLAGSPAIKKGILQTVKVVDELVKVMGR
                     HKPENIVIEMARENQTTQKGQKNSRERMKRIEEGIKELGSQILKEHPVENTQLQNEKLY
                     LYYLQNGRDMYVDQELDINRLSDYDVDAIVPQSFLKDDSIDNKVLTRSDKNRGKSDNVP
                     SEEVVKKMKNYWRQLLNAKLITQRKFDNLTKAERGGLSELDKAGFIKRQLVETRQITKH
                     VAQILDSRMNTKYDENDKLIREVKVITLKSKLVSDFRKDFQFYKVREINNYHHAHDAYL
                     NAVVGTALIKKYPKLESEFVYGDYKVYDVRKMIAKSEQEIGKATAKYFFYSNIMNFFKT
                     EITLANGEIRKRPLIETNGETGEIVWDKGRDFATVRKVLSMPQVNIVKKTEVQTGGFSK
                     ESILPKRNSDKLIARKKDWDPKKYGGFDSPTVAYSVLVVAKVEKGKSKKLKSVKELLGI
                     TIMERSSFEKNPIDFLEAKGYKEVKKDLIIKLPKYSLFELENGRKRMLASAGELQKGNE
                     LALPSKYVNFLYLASHYEKLKGSPEDNEQKQLFVEQHKHYLDEIIEQISEFSKRVILAD
                     ANLDKVLSAYNKHRDKPIREQAENIIHLFTLTNLGAPAAFKYFDTTIDRKRYTSTKEVL
                     DATLIHQSITGLYETRIDLSQLGGD"
     CDS             7516..7542
                     /codon_start=1
                     /product="HA (human influenza hemagglutinin) epitope tag"
                     /label=HA
                     /translation="YPYDVPDYA"
     CDS             7522..7548
                     /codon_start=1
                     /product="HA (human influenza hemagglutinin) epitope tag"
                     /label=HA
                     /note="This is a variant of the standard HA tag, which has 
                     sequence YPYDVPDYA. Some anti-HA antibodies may not 
                     recognize this variant."
                     /translation="YDVPDYASL"
     CDS             7561..7581
                     /codon_start=1
                     /product="nuclear localization signal of SV40 (simian virus
                     40) large T antigen"
                     /label=SV40 NLS
                     /translation="PKKKRKV"
     CDS             7588..7608
                     /codon_start=1
                     /product="nuclear localization signal of SV40 (simian virus
                     40) large T antigen"
                     /label=SV40 NLS
                     /translation="PKKKRKV"
     CDS             7651..7707
                     /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             7708..8304
                     /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     8201..8221
                     /label=Puro-F
                     /note="Puromycin resistance gene, forward primer"
     misc_feature    8320..8908
                     /label=WPRE
                     /note="woodchuck hepatitis virus posttranscriptional 
                     regulatory element"
     primer_bind     complement(8373..8393)
                     /label=WPRE-R
                     /note="WPRE, reverse primer"
     CDS             complement(8791..8802)
                     /codon_start=1
                     /product="Factor Xa recognition and cleavage site"
                     /label=Factor Xa site
                     /translation="IEGR"
     LTR             8980..9213
                     /label=3' LTR (Delta-U3)
                     /note="self-inactivating 3' long terminal repeat (LTR) from
                     HIV-1"
     primer_bind     complement(9239..9256)
                     /label=BGH-rev
                     /note="Bovine growth hormone terminator, reverse primer. 
                     Also called BGH reverse"
     polyA_signal    9245..9469
                     /label=bGH poly(A) signal
                     /note="bovine growth hormone polyadenylation signal"
     rep_origin      9515..9943
                     /direction=RIGHT
                     /label=f1 ori
                     /note="f1 bacteriophage origin of replication; arrow 
                     indicates direction of (+) strand synthesis"
     primer_bind     complement(9602..9621)
                     /label=F1ori-R
                     /note="F1 origin, reverse primer"
     primer_bind     9812..9833
                     /label=F1ori-F
                     /note="F1 origin, forward primer"
     primer_bind     complement(9952..9972)
                     /label=pBABE 3'
                     /note="SV40 enhancer, reverse primer for pBABE vectors"
     promoter        9957..10286
                     /label=SV40 promoter
                     /note="SV40 enhancer and early promoter"
     rep_origin      10137..10272
                     /label=SV40 ori
                     /note="SV40 origin of replication"
     primer_bind     10199..10218
                     /label=SV40pro-F
                     /note="SV40 promoter/origin, forward primer"
     promoter        10334..10381
                     /label=EM7 promoter
                     /note="synthetic bacterial promoter "
     CDS             10400..10774
                     /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    10904..11025
                     /label=SV40 poly(A) signal
                     /note="SV40 polyadenylation signal"
     primer_bind     complement(10941..10960)
                     /label=SV40pA-R
                     /note="SV40 polyA, reverse primer"
     primer_bind     10995..11014
                     /label=EBV-rev
                     /note="SV40 polyA terminator, reverse primer"
     primer_bind     complement(11074..11090)
                     /label=M13 rev
                     /note="common sequencing primer, one of multiple similar 
                     variants"
     primer_bind     complement(11074..11090)
                     /label=M13 Reverse
                     /note="In lacZ gene. Also called M13-rev"
     primer_bind     complement(11087..11109)
                     /label=M13/pUC Reverse
                     /note="In lacZ gene"
     protein_bind    11098..11114
                     /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(11122..11128,11129..11146,11147..11152))
                     /label=lac promoter
                     /note="promoter for the E. coli lac operon"
     protein_bind    11167..11188
                     /label=CAP binding site
                     /bound_moiety="E. coli catabolite activator protein"
                     /note="CAP binding activates transcription in the presence 
                     of cAMP."
     primer_bind     complement(11305..11322)
                     /label=L4440
                     /note="L4440 vector, forward primer"
     rep_origin      complement(11476..12064)
                     /direction=LEFT
                     /label=ori
                     /note="high-copy-number ColE1/pMB1/pBR322/pUC origin of 
                     replication"
     primer_bind     complement(11556..11575)
                     /label=pBR322ori-F
                     /note="pBR322 origin, forward primer"
     CDS             complement(join(12235..13026,13027..13095))
                     /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     12858..12877
                     /label=Amp-R
                     /note="Ampicillin resistance gene, reverse primer"
     promoter        complement(13096..13200)
                     /gene="bla"
                     /label=AmpR promoter
     primer_bind     complement(13275..13294)
                     /label=pRS-marker
                     /note="pRS vectors, use to sequence yeast selectable 
                     marker"
ORIGIN
        1 gaggtcgctg agtagtgcgc gagcaaaatt taagctacaa caaggcaagg cttgaccgac
       61 aattgcatga agaatctgct tagggttagg cgttttgcgc tgcttcgcga tgtacgggcc
      121 agatatacgc gttgacattg attattgact agttattaat agtaatcaat tacggggtca
      181 ttagttcata gcccatatat ggagttccgc gttacataac ttacggtaaa tggcccgcct
      241 ggctgaccgc ccaacgaccc ccgcccattg acgtcaataa tgacgtatgt tcccatagta
      301 acgccaatag ggactttcca ttgacgtcaa tgggtggagt atttacggta aactgcccac
      361 ttggcagtac atcaagtgta tcatatgcca agtacgcccc ctattgacgt caatgacggt
      421 aaatggcccg cctggcatta tgcccagtac atgaccttat gggactttcc tacttggcag
      481 tacatctacg tattagtcat cgctattacc atggtgatgc ggttttggca gtacatcaat
      541 gggcgtggat agcggtttga ctcacgggga tttccaagtc tccaccccat tgacgtcaat
      601 gggagtttgt tttggcacca aaatcaacgg gactttccaa aatgtcgtaa caactccgcc
      661 ccattgacgc aaatgggcgg taggcgtgta cggtgggagg tctatataag cagcgcgttt
      721 tgcctgtact gggtctctct ggttagacca gatctgagcc tgggagctct ctggctaact
      781 agggaaccca ctgcttaagc ctcaataaag cttgccttga gtgcttcaag tagtgtgtgc
      841 ccgtctgttg tgtgactctg gtaactagag atccctcaga cccttttagt cagtgtggaa
      901 aatctctagc agtggcgccc gaacagggac ttgaaagcga aagggaaacc agaggagctc
      961 tctcgacgca ggactcggct tgctgaagcg cgcacggcaa gaggcgaggg gcggcgactg
     1021 gtgagtacgc caaaaatttt gactagcgga ggctagaagg agagagatgg gtgcgagagc
     1081 gtcagtatta agcgggggag aattagatcg cgatgggaaa aaattcggtt aaggccaggg
     1141 ggaaagaaaa aatataaatt aaaacatata gtatgggcaa gcagggagct agaacgattc
     1201 gcagttaatc ctggcctgtt agaaacatca gaaggctgta gacaaatact gggacagcta
     1261 caaccatccc ttcagacagg atcagaagaa cttagatcat tatataatac agtagcaacc
     1321 ctctattgtg tgcatcaaag gatagagata aaagacacca aggaagcttt agacaagata
     1381 gaggaagagc aaaacaaaag taagaccacc gcacagcaag cggccgctga tcttcagacc
     1441 tggaggagga gatatgaggg acaattggag aagtgaatta tataaatata aagtagtaaa
     1501 aattgaacca ttaggagtag cacccaccaa ggcaaagaga agagtggtgc agagagaaaa
     1561 aagagcagtg ggaataggag ctttgttcct tgggttcttg ggagcagcag gaagcactat
     1621 gggcgcagcg tcaatgacgc tgacggtaca ggccagacaa ttattgtctg gtatagtgca
     1681 gcagcagaac aatttgctga gggctattga ggcgcaacag catctgttgc aactcacagt
     1741 ctggggcatc aagcagctcc aggcaagaat cctggctgtg gaaagatacc taaaggatca
     1801 acagctcctg gggatttggg gttgctctgg aaaactcatt tgcaccactg ctgtgccttg
     1861 gaatgctagt tggagtaata aatctctgga acagatttgg aatcacacga cctggatgga
     1921 gtgggacaga gaaattaaca attacacaag cttaatacac tccttaattg aagaatcgca
     1981 aaaccagcaa gaaaagaatg aacaagaatt attggaatta gataaatggg caagtttgtg
     2041 gaattggttt aacataacaa attggctgtg gtatataaaa ttattcataa tgatagtagg
     2101 aggcttggta ggtttaagaa tagtttttgc tgtactttct atagtgaata gagttaggca
     2161 gggatattca ccattatcgt ttcagaccca cctcccaacc ccgaggggac ccgacaggcc
     2221 cgaaggaata gaagaagaag gtggagagag agacagagac agatccattc gattagtgaa
     2281 cggatcggca ctgcgtgcgc caattctgca gacaaatggc agtattcatc cacaatttta
     2341 aaagaaaagg ggggattggg gggtacagtg caggggaaag aatagtagac ataatagcaa
     2401 cagacataca aactaaagaa ttacaaaaac aaattacaaa aattcaaaat tttcgggttt
     2461 attacaggga cagcagagat ccagtttggt taattaaggt accgagggcc tatttcccat
     2521 gattccttca tatttgcata tacgatacaa ggctgttaga gagataatta gaattaattt
     2581 gactgtaaac acaaagatat tagtacaaaa tacgtgacgt agaaagtaat aatttcttgg
     2641 gtagtttgca gttttaaaat tatgttttaa aatggactat catatgctta ccgtaacttg
     2701 aaagtatttc gatttcttgg ctttatatat cttgtggaaa ggacgaaaca ccggctgagc
     2761 gcaggccgga agagttttag agctagaaat agcaagttaa aataaggcta gtccgttatc
     2821 aacttgaaaa agtggcaccg agtcggtgct tttttgaatt cgctagctag gtcttgaaag
     2881 gagtgggaat tggctccggt gcccgtcagt gggcagagcg cacatcgccc acagtccccg
     2941 agaagttggg gggaggggtc ggcaattgat ccggtgccta gagaaggtgg cgcggggtaa
     3001 actgggaaag tgatgtcgtg tactggctcc gcctttttcc cgagggtggg ggagaaccgt
     3061 atataagtgc agtagtcgcc gtgaacgttc tttttcgcaa cgggtttgcc gccagaacac
     3121 aggaccggtt ctagcatcga ttgatcaaca agtttgtaca aaaaagttgg catggatgct
     3181 aagtcactaa ctgcctggtc ccggacactg gtgaccttca aggatgtatt tgtggacttc
     3241 accagggagg agtggaagct gctggacact gctcagcaga tcgtgtacag aaatgtgatg
     3301 ctggagaact ataagaacct ggtttccttg ggttatcagc ttactaagcc agatgtgatc
     3361 ctccggttgg agaagggaga agagcccggc ggttccggcg gagggtcgat ggacaagaag
     3421 tacagcatcg gcctggccat cggcaccaac tctgtgggct gggccgtgat caccgacgag
     3481 tacaaggtgc ccagcaagaa attcaaggtg ctgggcaaca ccgaccggca cagcatcaag
     3541 aagaacctga tcggcgccct gctgttcgac agcggagaaa cagccgaggc cacccggctg
     3601 aagagaaccg ccagaagaag atacaccaga cggaagaacc ggatctgcta tctgcaagag
     3661 atcttcagca acgagatggc caaggtggac gacagcttct tccacagact ggaagagtcc
     3721 ttcctggtgg aagaggataa gaagcacgag cggcacccca tcttcggcaa catcgtggac
     3781 gaggtggcct accacgagaa gtaccccacc atctaccacc tgagaaagaa actggtggac
     3841 agcaccgaca aggccgacct gcggctgatc tatctggccc tggcccacat gatcaagttc
     3901 cggggccact tcctgatcga gggcgacctg aaccccgaca acagcgacgt ggacaagctg
     3961 ttcatccagc tggtgcagac ctacaaccag ctgttcgagg aaaaccccat caacgccagc
     4021 ggcgtggacg ccaaggccat cctgtctgcc agactgagca agagcagacg gctggaaaat
     4081 ctgatcgccc agctgcccgg cgagaagaag aatggcctgt tcggcaacct gattgccctg
     4141 agcctgggcc tgacccccaa cttcaagagc aacttcgacc tggccgagga tgccaaactg
     4201 cagctgagca aggacaccta cgacgacgac ctggacaacc tgctggccca gatcggcgac
     4261 cagtacgccg acctgtttct ggccgccaag aacctgtccg acgccatcct gctgagcgac
     4321 atcctgagag tgaacaccga gatcaccaag gcccccctga gcgcctctat gatcaagaga
     4381 tacgacgagc accaccagga cctgaccctg ctgaaagctc tcgtgcggca gcagctgcct
     4441 gagaagtaca aagagatttt cttcgaccag agcaagaacg gctacgccgg ctacatcgat
     4501 ggcggagcca gccaggaaga gttctacaag ttcatcaagc ccatcctgga aaagatggac
     4561 ggcaccgagg aactgctcgt gaagctgaac agagaggacc tgctgcggaa gcagcggacc
     4621 ttcgacaacg gcagcatccc ccaccagatc cacctgggag agctgcacgc cattctgcgg
     4681 cggcaggaag atttttaccc attcctgaag gacaaccggg aaaagatcga gaagatcctg
     4741 accttccgca tcccctacta cgtgggccct ctggccaggg gaaacagcag attcgcctgg
     4801 atgaccagaa agagcgagga aaccatcacc ccctggaact tcgaggaagt ggtggacaag
     4861 ggcgccagcg cccagagctt catcgagcgg atgaccaact tcgataagaa cctgcccaac
     4921 gagaaggtgc tgcccaagca cagcctgctg tacgagtact tcaccgtgta caacgagctg
     4981 accaaagtga aatacgtgac cgagggaatg agaaagcccg ccttcctgag cggcgagcag
     5041 aaaaaagcca tcgtggacct gctgttcaag accaaccgga aagtgaccgt gaagcagctg
     5101 aaagaggact acttcaagaa aatcgagtgc ttcgactccg tggaaatctc cggcgtggaa
     5161 gatcggttca acgcctccct gggcacatac cacgatctgc tgaaaattat caaggacaag
     5221 gacttcctgg acaatgagga aaacgaggac attctggaag atatcgtgct gaccctgaca
     5281 ctgtttgagg acagagagat gatcgaggaa cggctgaaaa cctatgccca cctgttcgac
     5341 gacaaagtga tgaagcagct gaagcggcgg agatacaccg gctggggcag gctgagccgg
     5401 aagctgatca acggcatccg ggacaagcag tccggcaaga caatcctgga tttcctgaag
     5461 tccgacggct tcgccaacag aaacttcatg cagctgatcc acgacgacag cctgaccttt
     5521 aaagaggaca tccagaaagc ccaggtgtcc ggccagggcg atagcctgca cgagcacatt
     5581 gccaatctgg ccggcagccc cgccattaag aagggcatcc tgcagacagt gaaggtggtg
     5641 gacgagctcg tgaaagtgat gggccggcac aagcccgaga acatcgtgat cgaaatggcc
     5701 agagagaacc agaccaccca gaagggacag aagaacagcc gcgagagaat gaagcggatc
     5761 gaagagggca tcaaagagct gggcagccag atcctgaaag aacaccccgt ggaaaacacc
     5821 cagctgcaga acgagaagct gtacctgtac tacctgcaga atgggcggga tatgtacgtg
     5881 gaccaggaac tggacatcaa ccggctgtcc gactacgatg tggacgctat cgtgcctcag
     5941 agctttctga aggacgactc catcgataac aaagtgctga ctcggagcga caagaaccgg
     6001 ggcaagagcg acaacgtgcc ctccgaagag gtcgtgaaga agatgaagaa ctactggcgc
     6061 cagctgctga atgccaagct gattacccag aggaagttcg acaatctgac caaggccgag
     6121 agaggcggcc tgagcgaact ggataaggcc ggcttcatca agagacagct ggtggaaacc
     6181 cggcagatca caaagcacgt ggcacagatc ctggactccc ggatgaacac taagtacgac
     6241 gagaacgaca aactgatccg ggaagtgaaa gtgatcaccc tgaagtccaa gctggtgtcc
     6301 gatttccgga aggatttcca gttttacaaa gtgcgcgaga tcaacaacta ccaccacgcc
     6361 cacgacgcct acctgaacgc cgtcgtggga accgccctga tcaaaaagta ccctaagctg
     6421 gaaagcgagt tcgtgtacgg cgactacaag gtgtacgacg tgcggaagat gatcgccaag
     6481 agcgagcagg aaatcggcaa ggctaccgcc aagtacttct tctacagcaa catcatgaac
     6541 tttttcaaga ccgagattac cctggccaac ggcgagatcc ggaagcggcc tctgatcgag
     6601 acaaacggcg aaacaggcga gatcgtgtgg gataagggcc gggactttgc caccgtgcgg
     6661 aaagtgctgt ctatgcccca agtgaatatc gtgaaaaaga ccgaggtgca gacaggcggc
     6721 ttcagcaaag agtctatcct gcccaagagg aacagcgaca agctgatcgc cagaaagaag
     6781 gactgggacc ctaagaagta cggcggcttc gacagcccca ccgtggccta ttctgtgctg
     6841 gtggtggcca aagtggaaaa gggcaagtcc aagaaactga agagtgtgaa agagctgctg
     6901 gggatcacca tcatggaaag aagcagcttc gagaagaatc ccatcgactt tctggaagcc
     6961 aagggctaca aagaagtgaa aaaggacctg atcatcaagc tgcctaagta ctccctgttc
     7021 gagctggaaa acggccggaa gagaatgctg gcctctgccg gcgaactgca gaagggaaac
     7081 gaactggccc tgccctccaa atatgtgaac ttcctgtacc tggccagcca ctatgagaag
     7141 ctgaagggct cccccgagga taatgagcag aaacagctgt ttgtggaaca gcacaaacac
     7201 tacctggacg agatcatcga gcagatcagc gagttctcca agagagtgat cctggccgac
     7261 gctaatctgg acaaggtgct gagcgcctac aacaagcaca gagacaagcc tatcagagag
     7321 caggccgaga atatcatcca cctgtttacc ctgaccaatc tgggagcccc tgccgccttc
     7381 aagtactttg acaccaccat cgaccggaag aggtacacca gcaccaaaga ggtgctggac
     7441 gccaccctga tccaccagag catcaccggc ctgtacgaga cacggatcga cctgtctcag
     7501 ctgggaggcg acgcctatcc ctatgacgtg cccgattatg ccagcctggg cagcggctcc
     7561 cccaagaaaa aacgcaaggt ggaagatcct aagaaaaagc ggaaagtgga cggcattggt
     7621 agtgggagca acggcagcag cggatccggc gcaacaaact tctctctgct gaaacaagcc
     7681 ggagatgtcg aagagaatcc tggaccgacc gagtacaagc ccacggtgcg cctcgccacc
     7741 cgcgacgacg tccccagggc cgtacgcacc ctcgccgccg cgttcgccga ctaccccgcc
     7801 acgcgccaca ccgtcgatcc ggaccgccac atcgagcggg tcaccgagct gcaagaactc
     7861 ttcctcacgc gcgtcgggct cgacatcggc aaggtgtggg tcgcggacga cggcgccgcg
     7921 gtggcggtct ggaccacgcc ggagagcgtc gaagcggggg cggtgttcgc cgagatcggc
     7981 ccgcgcatgg ccgagttgag cggttcccgg ctggccgcgc agcaacagat ggaaggcctc
     8041 ctggcgccgc accggcccaa ggagcccgcg tggttcctgg ccaccgtcgg agtctcgccc
     8101 gaccaccagg gcaagggtct gggcagcgcc gtcgtgctcc ccggagtgga ggcggccgag
     8161 cgcgccgggg tgcccgcctt cctggagacc tccgcgcccc gcaacctccc cttctacgag
     8221 cggctcggct tcaccgtcac cgccgacgtc gaggtgcccg aaggaccgcg cacctggtgc
     8281 atgacccgca agcccggtgc ctgaacgcgt taagtcgaca atcaacctct ggattacaaa
     8341 atttgtgaaa gattgactgg tattcttaac tatgttgctc cttttacgct atgtggatac
     8401 gctgctttaa tgcctttgta tcatgctatt gcttcccgta tggctttcat tttctcctcc
     8461 ttgtataaat cctggttgct gtctctttat gaggagttgt ggcccgttgt caggcaacgt
     8521 ggcgtggtgt gcactgtgtt tgctgacgca acccccactg gttggggcat tgccaccacc
     8581 tgtcagctcc tttccgggac tttcgctttc cccctcccta ttgccacggc ggaactcatc
     8641 gccgcctgcc ttgcccgctg ctggacaggg gctcggctgt tgggcactga caattccgtg
     8701 gtgttgtcgg ggaaatcatc gtcctttcct tggctgctcg cctgtgttgc cacctggatt
     8761 ctgcgcggga cgtccttctg ctacgtccct tcggccctca atccagcgga ccttccttcc
     8821 cgcggcctgc tgccggctct gcggcctctt ccgcgtcttc gccttcgccc tcagacgagt
     8881 cggatctccc tttgggccgc ctccccgcgt cgactttaag accaatgact tacaaggcag
     8941 ctgtagatct tagccacttt ttaaaagaaa aggggggact ggaagggcta attcactccc
     9001 aacgaagaca agatctgctt tttgcttgta ctgggtctct ctggttagac cagatctgag
     9061 cctgggagct ctctggctaa ctagggaacc cactgcttaa gcctcaataa agcttgcctt
     9121 gagtgcttca agtagtgtgt gcccgtctgt tgtgtgactc tggtaactag agatccctca
     9181 gaccctttta gtcagtgtgg aaaatctcta gcagggcccg tttaaacccg ctgatcagcc
     9241 tcgactgtgc cttctagttg ccagccatct gttgtttgcc cctcccccgt gccttccttg
     9301 accctggaag gtgccactcc cactgtcctt tcctaataaa atgaggaaat tgcatcgcat
     9361 tgtctgagta ggtgtcattc tattctgggg ggtggggtgg ggcaggacag caagggggag
     9421 gattgggaag acaatagcag gcatgctggg gatgcggtgg gctctatggc ttctgaggcg
     9481 gaaagaacca gctggggctc tagggggtat ccccacgcgc cctgtagcgg cgcattaagc
     9541 gcggcgggtg tggtggttac gcgcagcgtg accgctacac ttgccagcgc cctagcgccc
     9601 gctcctttcg ctttcttccc ttcctttctc gccacgttcg ccggctttcc ccgtcaagct
     9661 ctaaatcggg ggctcccttt agggttccga tttagtgctt tacggcacct cgaccccaaa
     9721 aaacttgatt agggtgatgg ttcacgtagt gggccatcgc cctgatagac ggtttttcgc
     9781 cctttgacgt tggagtccac gttctttaat agtggactct tgttccaaac tggaacaaca
     9841 ctcaacccta tctcggtcta ttcttttgat ttataaggga ttttgccgat ttcggcctat
     9901 tggttaaaaa atgagctgat ttaacaaaaa tttaacgcga attaattctg tggaatgtgt
     9961 gtcagttagg gtgtggaaag tccccaggct ccccagcagg cagaagtatg caaagcatgc
    10021 atctcaatta gtcagcaacc aggtgtggaa agtccccagg ctccccagca ggcagaagta
    10081 tgcaaagcat gcatctcaat tagtcagcaa ccatagtccc gcccctaact ccgcccatcc
    10141 cgcccctaac tccgcccagt tccgcccatt ctccgcccca tggctgacta atttttttta
    10201 tttatgcaga ggccgaggcc gcctctgcct ctgagctatt ccagaagtag tgaggaggct
    10261 tttttggagg cctaggcttt tgcaaaaagc tcccgggagc ttgtatatcc attttcggat
    10321 ctgatcagca cgtgttgaca attaatcatc ggcatagtat atcggcatag tataatacga
    10381 caaggtgagg aactaaacca tggccaagtt gaccagtgcc gttccggtgc tcaccgcgcg
    10441 cgacgtcgcc ggagcggtcg agttctggac cgaccggctc gggttctccc gggacttcgt
    10501 ggaggacgac ttcgccggtg tggtccggga cgacgtgacc ctgttcatca gcgcggtcca
    10561 ggaccaggtg gtgccggaca acaccctggc ctgggtgtgg gtgcgcggcc tggacgagct
    10621 gtacgccgag tggtcggagg tcgtgtccac gaacttccgg gacgcctccg ggccggccat
    10681 gaccgagatc ggcgagcagc cgtgggggcg ggagttcgcc ctgcgcgacc cggccggcaa
    10741 ctgcgtgcac ttcgtggccg aggagcagga ctgacacgtg ctacgagatt tcgattccac
    10801 cgccgccttc tatgaaaggt tgggcttcgg aatcgttttc cgggacgccg gctggatgat
    10861 cctccagcgc ggggatctca tgctggagtt cttcgcccac cccaacttgt ttattgcagc
    10921 ttataatggt tacaaataaa gcaatagcat cacaaatttc acaaataaag catttttttc
    10981 actgcattct agttgtggtt tgtccaaact catcaatgta tcttatcatg tctgtatacc
    11041 gtcgacctct agctagagct tggcgtaatc atggtcatag ctgtttcctg tgtgaaattg
    11101 ttatccgctc acaattccac acaacatacg agccggaagc ataaagtgta aagcctgggg
    11161 tgcctaatga gtgagctaac tcacattaat tgcgttgcgc tcactgcccg ctttccagtc
    11221 gggaaacctg tcgtgccagc tgcattaatg aatcggccaa cgcgcgggga gaggcggttt
    11281 gcgtattggg cgctcttccg cttcctcgct cactgactcg ctgcgctcgg tcgttcggct
    11341 gcggcgagcg gtatcagctc actcaaaggc ggtaatacgg ttatccacag aatcagggga
    11401 taacgcagga aagaacatgt gagcaaaagg ccagcaaaag gccaggaacc gtaaaaaggc
    11461 cgcgttgctg gcgtttttcc ataggctccg cccccctgac gagcatcaca aaaatcgacg
    11521 ctcaagtcag aggtggcgaa acccgacagg actataaaga taccaggcgt ttccccctgg
    11581 aagctccctc gtgcgctctc ctgttccgac cctgccgctt accggatacc tgtccgcctt
    11641 tctcccttcg ggaagcgtgg cgctttctca tagctcacgc tgtaggtatc tcagttcggt
    11701 gtaggtcgtt cgctccaagc tgggctgtgt gcacgaaccc cccgttcagc ccgaccgctg
    11761 cgccttatcc ggtaactatc gtcttgagtc caacccggta agacacgact tatcgccact
    11821 ggcagcagcc actggtaaca ggattagcag agcgaggtat gtaggcggtg ctacagagtt
    11881 cttgaagtgg tggcctaact acggctacac tagaagaaca gtatttggta tctgcgctct
    11941 gctgaagcca gttaccttcg gaaaaagagt tggtagctct tgatccggca aacaaaccac
    12001 cgctggtagc ggtggttttt ttgtttgcaa gcagcagatt acgcgcagaa aaaaaggatc
    12061 tcaagaagat cctttgatct tttctacggg gtctgacgct cagtggaacg aaaactcacg
    12121 ttaagggatt ttggtcatga gattatcaaa aaggatcttc acctagatcc ttttaaatta
    12181 aaaatgaagt tttaaatcaa tctaaagtat atatgagtaa acttggtctg acagttacca
    12241 atgcttaatc agtgaggcac ctatctcagc gatctgtcta tttcgttcat ccatagttgc
    12301 ctgactcccc gtcgtgtaga taactacgat acgggagggc ttaccatctg gccccagtgc
    12361 tgcaatgata ccgcgagacc cacgctcacc ggctccagat ttatcagcaa taaaccagcc
    12421 agccggaagg gccgagcgca gaagtggtcc tgcaacttta tccgcctcca tccagtctat
    12481 taattgttgc cgggaagcta gagtaagtag ttcgccagtt aatagtttgc gcaacgttgt
    12541 tgccattgct acaggcatcg tggtgtcacg ctcgtcgttt ggtatggctt cattcagctc
    12601 cggttcccaa cgatcaaggc gagttacatg atcccccatg ttgtgcaaaa aagcggttag
    12661 ctccttcggt cctccgatcg ttgtcagaag taagttggcc gcagtgttat cactcatggt
    12721 tatggcagca ctgcataatt ctcttactgt catgccatcc gtaagatgct tttctgtgac
    12781 tggtgagtac tcaaccaagt cattctgaga atagtgtatg cggcgaccga gttgctcttg
    12841 cccggcgtca atacgggata ataccgcgcc acatagcaga actttaaaag tgctcatcat
    12901 tggaaaacgt tcttcggggc gaaaactctc aaggatctta ccgctgttga gatccagttc
    12961 gatgtaaccc actcgtgcac ccaactgatc ttcagcatct tttactttca ccagcgtttc
    13021 tgggtgagca aaaacaggaa ggcaaaatgc cgcaaaaaag ggaataaggg cgacacggaa
    13081 atgttgaata ctcatactct tcctttttca atattattga agcatttatc agggttattg
    13141 tctcatgagc ggatacatat ttgaatgtat ttagaaaaat aaacaaatag gggttccgcg
    13201 cacatttccc cgaaaagtgc cacctgacgt cgacggatcg ggagatctcc cgatccccta
    13261 tggtgcactc tcagtacaat ctgctctgat gccgcatagt taagccagta tctgctccct
    13321 gcttgtgtgt tg
//
