LOCUS       sequence-273051-       13212 bp    DNA     circular SYN 15-DEC-2025
DEFINITION  Expresses a human CRTC2-targeting sgRNA and Cas9.
ACCESSION   .
VERSION     .
KEYWORDS    .
SOURCE      synthetic DNA construct
  ORGANISM  synthetic DNA construct
REFERENCE   1  (bases 1 to 13212)
  AUTHORS   Hollstein PE, Eichner LJ, Brun SN, Kamireddy A, Svensson RU, Vera 
            LI, Ross DS, Rymoff TJ, Hutchins A, Galvez HM, Williams AE, 
            Shokhirev MN, Screaton RA, Berdeaux R, Shaw RJ
  TITLE     The AMPK-Related Kinases SIK1 and SIK3 Mediate Key Tumor-Suppressive
            Effects of LKB1 in NSCLC.
  JOURNAL   Cancer Discov. 2019 Nov;9(11):1606-1627. doi: 
            10.1158/2159-8290.CD-18-1261. Epub 2019 Jul 26.
  PUBMED    31350328
REFERENCE   2  (bases 1 to 13212)
  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.CD-18-1261"; journalName: "Cancer Discov"; date: 
            "2019-11"; volume: "9"; issue: "11"; pages: "1606-1627"
COMMENT     Sequence Label: pLenti-Neo-sgCRTC2-2
FEATURES             Location/Qualifiers
     source          1..13212
                     /mol_type="other DNA"
                     /organism="synthetic DNA construct"
     promoter        1..199
                     /label=CMV promoter
                     /note="human cytomegalovirus (CMV) immediate early 
                     promoter"
     primer_bind     154..174
                     /label=CMV-F
                     /note="Human CMV immediate early promoter, forward primer"
     LTR             217..397
                     /label=5' LTR (truncated)
                     /note="truncated 5' long terminal repeat (LTR) from HIV-1"
     misc_feature    444..569
                     /label=HIV-1 Psi
                     /note="packaging signal of human immunodeficiency virus 
                     type 1"
     misc_feature    1062..1295
                     /label=RRE
                     /note="The Rev response element (RRE) of HIV-1 allows for 
                     Rev-dependent mRNA export from the nucleus to the 
                     cytoplasm."
     CDS             1480..1524
                     /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    1822..1939
                     /label=cPPT/CTS
                     /note="central polypurine tract and central termination 
                     sequence of HIV-1"
     promoter        1990..2238
                     /label=U6 promoter
                     /note="RNA polymerase III promoter for human U6 snRNA 
                     (Domitrovich & Kunkel, 2003)"
     primer_bind     1990..2010
                     /label=hU6-F
                     /note="Human U6 promoter, forward primer"
     primer_bind     2161..2180
                     /label=LKO.1 5'
                     /note="Human U6 promoter, forward primer"
     misc_RNA        2260..2335
                     /label=gRNA scaffold
                     /note="guide RNA scaffold for the Streptococcus pyogenes 
                     CRISPR/Cas9 system"
     promoter        2397..2608
                     /label=EF-1-alpha core promoter
                     /note="core promoter for human elongation factor 
                     EF-1-alpha"
     regulatory      2627..2636
                     /label=Kozak sequence
                     /note="vertebrate consensus sequence for strong initiation 
                     of translation (Kozak, 1987)"
                     /regulatory_class="other"
     CDS             2633..6736
                     /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             2633..6736
                     /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             6737..6784
                     /codon_start=1
                     /product="bipartite nuclear localization signal from 
                     nucleoplasmin"
                     /label=nucleoplasmin NLS
                     /translation="KRPAATKKAGQAKKKK"
     CDS             6785..6808
                     /codon_start=1
                     /product="FLAG(R) epitope tag, followed by an enterokinase 
                     cleavage site"
                     /label=FLAG
                     /translation="DYKDDDDK"
     CDS             6818..6874
                     /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             6875..7666
                     /codon_start=1
                     /gene="aph(3')-II (or nptII)"
                     /product="aminoglycoside phosphotransferase from Tn5"
                     /label=NeoR/KanR
                     /note="confers resistance to neomycin, kanamycin, and G418 
                     (Geneticin(R))"
                     /translation="IEQDGLHAGSPAAWVERLFGYDWAQQTIGCSDAAVFRLSAQGRPV
                     LFVKTDLSGALNELQDEAARLSWLATTGVPCAAVLDVVTEAGRDWLLLGEVPGQDLLSS
                     HLAPAEKVSIMADAMRRLHTLDPATCPFDHQAKHRIERARTRMEAGLVDQDDLDEEHQG
                     LAPAELFARLKARMPDGEDLVVTHGDACLPNIMVENGRFSGFIDCGRLGVADRYQDIAL
                     ATRDIAEELGGEWADRFLVLYGIAAPDSQRIAFYRLLDEFF"
     primer_bind     complement(6926..6945)
                     /label=Neo-R
                     /note="Neomycin resistance gene, reverse primer"
     primer_bind     7536..7555
                     /label=Neo-F
                     /note="Neomycin resistance gene, forward primer"
     misc_feature    7682..8270
                     /label=WPRE
                     /note="woodchuck hepatitis virus posttranscriptional 
                     regulatory element"
     primer_bind     complement(7735..7755)
                     /label=WPRE-R
                     /note="WPRE, reverse primer"
     CDS             complement(8153..8164)
                     /codon_start=1
                     /product="Factor Xa recognition and cleavage site"
                     /label=Factor Xa site
                     /translation="IEGR"
     LTR             8342..8575
                     /label=3' LTR (Delta-U3)
                     /note="self-inactivating 3' long terminal repeat (LTR) from
                     HIV-1"
     primer_bind     complement(8601..8618)
                     /label=BGH-rev
                     /note="Bovine growth hormone terminator, reverse primer. 
                     Also called BGH reverse"
     polyA_signal    8607..8831
                     /label=bGH poly(A) signal
                     /note="bovine growth hormone polyadenylation signal"
     rep_origin      8877..9305
                     /direction=RIGHT
                     /label=f1 ori
                     /note="f1 bacteriophage origin of replication; arrow 
                     indicates direction of (+) strand synthesis"
     primer_bind     complement(8964..8983)
                     /label=F1ori-R
                     /note="F1 origin, reverse primer"
     primer_bind     9174..9195
                     /label=F1ori-F
                     /note="F1 origin, forward primer"
     primer_bind     complement(9314..9334)
                     /label=pBABE 3'
                     /note="SV40 enhancer, reverse primer for pBABE vectors"
     promoter        9319..9648
                     /label=SV40 promoter
                     /note="SV40 enhancer and early promoter"
     rep_origin      9499..9634
                     /label=SV40 ori
                     /note="SV40 origin of replication"
     primer_bind     9561..9580
                     /label=SV40pro-F
                     /note="SV40 promoter/origin, forward primer"
     promoter        9696..9743
                     /label=EM7 promoter
                     /note="synthetic bacterial promoter "
     CDS             9762..10136
                     /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    10266..10387
                     /label=SV40 poly(A) signal
                     /note="SV40 polyadenylation signal"
     primer_bind     complement(10303..10322)
                     /label=SV40pA-R
                     /note="SV40 polyA, reverse primer"
     primer_bind     10357..10376
                     /label=EBV-rev
                     /note="SV40 polyA terminator, reverse primer"
     primer_bind     complement(10436..10452)
                     /label=M13 rev
                     /note="common sequencing primer, one of multiple similar 
                     variants"
     primer_bind     complement(10436..10452)
                     /label=M13 Reverse
                     /note="In lacZ gene. Also called M13-rev"
     primer_bind     complement(10449..10471)
                     /label=M13/pUC Reverse
                     /note="In lacZ gene"
     protein_bind    10460..10476
                     /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(10484..10490,10491..10508,10509..10514))
                     /label=lac promoter
                     /note="promoter for the E. coli lac operon"
     protein_bind    10529..10550
                     /label=CAP binding site
                     /bound_moiety="E. coli catabolite activator protein"
                     /note="CAP binding activates transcription in the presence 
                     of cAMP."
     primer_bind     complement(10667..10684)
                     /label=L4440
                     /note="L4440 vector, forward primer"
     rep_origin      complement(10838..11426)
                     /direction=LEFT
                     /label=ori
                     /note="high-copy-number ColE1/pMB1/pBR322/pUC origin of 
                     replication"
     primer_bind     complement(10918..10937)
                     /label=pBR322ori-F
                     /note="pBR322 origin, forward primer"
     CDS             complement(join(11597..12388,12389..12457))
                     /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     12220..12239
                     /label=Amp-R
                     /note="Ampicillin resistance gene, reverse primer"
     promoter        complement(12458..12562)
                     /gene="bla"
                     /label=AmpR promoter
     primer_bind     complement(12637..12656)
                     /label=pRS-marker
                     /note="pRS vectors, use to sequence yeast selectable 
                     marker"
     enhancer        12832..13211
                     /label=CMV enhancer
                     /note="human cytomegalovirus immediate early enhancer"
ORIGIN
        1 tgatgcggtt ttggcagtac atcaatgggc gtggatagcg gtttgactca cggggatttc
       61 caagtctcca ccccattgac gtcaatggga gtttgttttg gcaccaaaat caacgggact
      121 ttccaaaatg tcgtaacaac tccgccccat tgacgcaaat gggcggtagg cgtgtacggt
      181 gggaggtcta tataagcagc gcgttttgcc tgtactgggt ctctctggtt agaccagatc
      241 tgagcctggg agctctctgg ctaactaggg aacccactgc ttaagcctca ataaagcttg
      301 ccttgagtgc ttcaagtagt gtgtgcccgt ctgttgtgtg actctggtaa ctagagatcc
      361 ctcagaccct tttagtcagt gtggaaaatc tctagcagtg gcgcccgaac agggacttga
      421 aagcgaaagg gaaaccagag gagctctctc gacgcaggac tcggcttgct gaagcgcgca
      481 cggcaagagg cgaggggcgg cgactggtga gtacgccaaa aattttgact agcggaggct
      541 agaaggagag agatgggtgc gagagcgtca gtattaagcg ggggagaatt agatcgcgat
      601 gggaaaaaat tcggttaagg ccagggggaa agaaaaaata taaattaaaa catatagtat
      661 gggcaagcag ggagctagaa cgattcgcag ttaatcctgg cctgttagaa acatcagaag
      721 gctgtagaca aatactggga cagctacaac catcccttca gacaggatca gaagaactta
      781 gatcattata taatacagta gcaaccctct attgtgtgca tcaaaggata gagataaaag
      841 acaccaagga agctttagac aagatagagg aagagcaaaa caaaagtaag accaccgcac
      901 agcaagcggc cgctgatctt cagacctgga ggaggagata tgagggacaa ttggagaagt
      961 gaattatata aatataaagt agtaaaaatt gaaccattag gagtagcacc caccaaggca
     1021 aagagaagag tggtgcagag agaaaaaaga gcagtgggaa taggagcttt gttccttggg
     1081 ttcttgggag cagcaggaag cactatgggc gcagcgtcaa tgacgctgac ggtacaggcc
     1141 agacaattat tgtctggtat agtgcagcag cagaacaatt tgctgagggc tattgaggcg
     1201 caacagcatc tgttgcaact cacagtctgg ggcatcaagc agctccaggc aagaatcctg
     1261 gctgtggaaa gatacctaaa ggatcaacag ctcctgggga tttggggttg ctctggaaaa
     1321 ctcatttgca ccactgctgt gccttggaat gctagttgga gtaataaatc tctggaacag
     1381 atttggaatc acacgacctg gatggagtgg gacagagaaa ttaacaatta cacaagctta
     1441 atacactcct taattgaaga atcgcaaaac cagcaagaaa agaatgaaca agaattattg
     1501 gaattagata aatgggcaag tttgtggaat tggtttaaca taacaaattg gctgtggtat
     1561 ataaaattat tcataatgat agtaggaggc ttggtaggtt taagaatagt ttttgctgta
     1621 ctttctatag tgaatagagt taggcaggga tattcaccat tatcgtttca gacccacctc
     1681 ccaaccccga ggggacccga caggcccgaa ggaatagaag aagaaggtgg agagagagac
     1741 agagacagat ccattcgatt agtgaacgga tcggcactgc gtgcgccaat tctgcagaca
     1801 aatggcagta ttcatccaca attttaaaag aaaagggggg attggggggt acagtgcagg
     1861 ggaaagaata gtagacataa tagcaacaga catacaaact aaagaattac aaaaacaaat
     1921 tacaaaaatt caaaattttc gggtttatta cagggacagc agagatccag tttggttaat
     1981 taaggtaccg agggcctatt tcccatgatt ccttcatatt tgcatatacg atacaaggct
     2041 gttagagaga taattagaat taatttgact gtaaacacaa agatattagt acaaaatacg
     2101 tgacgtagaa agtaataatt tcttgggtag tttgcagttt taaaattatg ttttaaaatg
     2161 gactatcata tgcttaccgt aacttgaaag tatttcgatt tcttggcttt atatatcttg
     2221 tggaaaggac gaaacaccga caggcccaaa aactgcgacg ttttagagct agaaatagca
     2281 agttaaaata aggctagtcc gttatcaact tgaaaaagtg gcaccgagtc ggtgcttttt
     2341 tgaattcgct agctaggtct tgaaaggagt gggaattggc tccggtgccc gtcagtgggc
     2401 agagcgcaca tcgcccacag tccccgagaa gttgggggga ggggtcggca attgatccgg
     2461 tgcctagaga aggtggcgcg gggtaaactg ggaaagtgat gtcgtgtact ggctccgcct
     2521 ttttcccgag ggtgggggag aaccgtatat aagtgcagta gtcgccgtga acgttctttt
     2581 tcgcaacggg tttgccgcca gaacacagga ccggttctag agcgctgcca ccatggacaa
     2641 gaagtacagc atcggcctgg acatcggcac caactctgtg ggctgggccg tgatcaccga
     2701 cgagtacaag gtgcccagca agaaattcaa ggtgctgggc aacaccgacc ggcacagcat
     2761 caagaagaac ctgatcggag ccctgctgtt cgacagcggc gaaacagccg aggccacccg
     2821 gctgaagaga accgccagaa gaagatacac cagacggaag aaccggatct gctatctgca
     2881 agagatcttc agcaacgaga tggccaaggt ggacgacagc ttcttccaca gactggaaga
     2941 gtccttcctg gtggaagagg ataagaagca cgagcggcac cccatcttcg gcaacatcgt
     3001 ggacgaggtg gcctaccacg agaagtaccc caccatctac cacctgagaa agaaactggt
     3061 ggacagcacc gacaaggccg acctgcggct gatctatctg gccctggccc acatgatcaa
     3121 gttccggggc cacttcctga tcgagggcga cctgaacccc gacaacagcg acgtggacaa
     3181 gctgttcatc cagctggtgc agacctacaa ccagctgttc gaggaaaacc ccatcaacgc
     3241 cagcggcgtg gacgccaagg ccatcctgtc tgccagactg agcaagagca gacggctgga
     3301 aaatctgatc gcccagctgc ccggcgagaa gaagaatggc ctgttcggaa acctgattgc
     3361 cctgagcctg ggcctgaccc ccaacttcaa gagcaacttc gacctggccg aggatgccaa
     3421 actgcagctg agcaaggaca cctacgacga cgacctggac aacctgctgg cccagatcgg
     3481 cgaccagtac gccgacctgt ttctggccgc caagaacctg tccgacgcca tcctgctgag
     3541 cgacatcctg agagtgaaca ccgagatcac caaggccccc ctgagcgcct ctatgatcaa
     3601 gagatacgac gagcaccacc aggacctgac cctgctgaaa gctctcgtgc ggcagcagct
     3661 gcctgagaag tacaaagaga ttttcttcga ccagagcaag aacggctacg ccggctacat
     3721 tgacggcgga gccagccagg aagagttcta caagttcatc aagcccatcc tggaaaagat
     3781 ggacggcacc gaggaactgc tcgtgaagct gaacagagag gacctgctgc ggaagcagcg
     3841 gaccttcgac aacggcagca tcccccacca gatccacctg ggagagctgc acgccattct
     3901 gcggcggcag gaagattttt acccattcct gaaggacaac cgggaaaaga tcgagaagat
     3961 cctgaccttc cgcatcccct actacgtggg ccctctggcc aggggaaaca gcagattcgc
     4021 ctggatgacc agaaagagcg aggaaaccat caccccctgg aacttcgagg aagtggtgga
     4081 caagggcgct tccgcccaga gcttcatcga gcggatgacc aacttcgata agaacctgcc
     4141 caacgagaag gtgctgccca agcacagcct gctgtacgag tacttcaccg tgtataacga
     4201 gctgaccaaa gtgaaatacg tgaccgaggg aatgagaaag cccgccttcc tgagcggcga
     4261 gcagaaaaag gccatcgtgg acctgctgtt caagaccaac cggaaagtga ccgtgaagca
     4321 gctgaaagag gactacttca agaaaatcga gtgcttcgac tccgtggaaa tctccggcgt
     4381 ggaagatcgg ttcaacgcct ccctgggcac ataccacgat ctgctgaaaa ttatcaagga
     4441 caaggacttc ctggacaatg aggaaaacga ggacattctg gaagatatcg tgctgaccct
     4501 gacactgttt gaggacagag agatgatcga ggaacggctg aaaacctatg cccacctgtt
     4561 cgacgacaaa gtgatgaagc agctgaagcg gcggagatac accggctggg gcaggctgag
     4621 ccggaagctg atcaacggca tccgggacaa gcagtccggc aagacaatcc tggatttcct
     4681 gaagtccgac ggcttcgcca acagaaactt catgcagctg atccacgacg acagcctgac
     4741 ctttaaagag gacatccaga aagcccaggt gtccggccag ggcgatagcc tgcacgagca
     4801 cattgccaat ctggccggca gccccgccat taagaagggc atcctgcaga cagtgaaggt
     4861 ggtggacgag ctcgtgaaag tgatgggccg gcacaagccc gagaacatcg tgatcgaaat
     4921 ggccagagag aaccagacca cccagaaggg acagaagaac agccgcgaga gaatgaagcg
     4981 gatcgaagag ggcatcaaag agctgggcag ccagatcctg aaagaacacc ccgtggaaaa
     5041 cacccagctg cagaacgaga agctgtacct gtactacctg cagaatgggc gggatatgta
     5101 cgtggaccag gaactggaca tcaaccggct gtccgactac gatgtggacc atatcgtgcc
     5161 tcagagcttt ctgaaggacg actccatcga caacaaggtg ctgaccagaa gcgacaagaa
     5221 ccggggcaag agcgacaacg tgccctccga agaggtcgtg aagaagatga agaactactg
     5281 gcggcagctg ctgaacgcca agctgattac ccagagaaag ttcgacaatc tgaccaaggc
     5341 cgagagaggc ggcctgagcg aactggataa ggccggcttc atcaagagac agctggtgga
     5401 aacccggcag atcacaaagc acgtggcaca gatcctggac tcccggatga acactaagta
     5461 cgacgagaat gacaagctga tccgggaagt gaaagtgatc accctgaagt ccaagctggt
     5521 gtccgatttc cggaaggatt tccagtttta caaagtgcgc gagatcaaca actaccacca
     5581 cgcccacgac gcctacctga acgccgtcgt gggaaccgcc ctgatcaaaa agtaccctaa
     5641 gctggaaagc gagttcgtgt acggcgacta caaggtgtac gacgtgcgga agatgatcgc
     5701 caagagcgag caggaaatcg gcaaggctac cgccaagtac ttcttctaca gcaacatcat
     5761 gaactttttc aagaccgaga ttaccctggc caacggcgag atccggaagc ggcctctgat
     5821 cgagacaaac ggcgaaaccg gggagatcgt gtgggataag ggccgggatt ttgccaccgt
     5881 gcggaaagtg ctgagcatgc cccaagtgaa tatcgtgaaa aagaccgagg tgcagacagg
     5941 cggcttcagc aaagagtcta tcctgcccaa gaggaacagc gataagctga tcgccagaaa
     6001 gaaggactgg gaccctaaga agtacggcgg cttcgacagc cccaccgtgg cctattctgt
     6061 gctggtggtg gccaaagtgg aaaagggcaa gtccaagaaa ctgaagagtg tgaaagagct
     6121 gctggggatc accatcatgg aaagaagcag cttcgagaag aatcccatcg actttctgga
     6181 agccaagggc tacaaagaag tgaaaaagga cctgatcatc aagctgccta agtactccct
     6241 gttcgagctg gaaaacggcc ggaagagaat gctggcctct gccggcgaac tgcagaaggg
     6301 aaacgaactg gccctgccct ccaaatatgt gaacttcctg tacctggcca gccactatga
     6361 gaagctgaag ggctcccccg aggataatga gcagaaacag ctgtttgtgg aacagcacaa
     6421 gcactacctg gacgagatca tcgagcagat cagcgagttc tccaagagag tgatcctggc
     6481 cgacgctaat ctggacaaag tgctgtccgc ctacaacaag caccgggata agcccatcag
     6541 agagcaggcc gagaatatca tccacctgtt taccctgacc aatctgggag cccctgccgc
     6601 cttcaagtac tttgacacca ccatcgaccg gaagaggtac accagcacca aagaggtgct
     6661 ggacgccacc ctgatccacc agagcatcac cggcctgtac gagacacgga tcgacctgtc
     6721 tcagctggga ggcgacaagc gacctgccgc cacaaagaag gctggacagg ctaagaagaa
     6781 gaaagattac aaagacgatg acgataaggg atccggcgca acaaacttct ctctgctgaa
     6841 acaagccgga gatgtcgaag agaatcctgg accgattgaa caagatggat tgcacgcagg
     6901 ttctccggcc gcttgggtgg agaggctatt cggctatgac tgggcacaac agacaatcgg
     6961 ctgctctgat gccgccgtgt tccggctgtc agcgcagggg cgcccggttc tttttgtcaa
     7021 gaccgacctg tccggtgccc tgaatgaact gcaggacgag gcagcgcggc tatcgtggct
     7081 ggccacgacg ggcgttcctt gcgcagctgt gctcgacgtt gtcactgaag cgggaaggga
     7141 ctggctgcta ttgggcgaag tgccggggca ggatctcctg tcatctcacc ttgctcctgc
     7201 cgagaaagta tccatcatgg ctgatgcaat gcggcggctg catacgcttg atccggctac
     7261 ctgcccattc gaccaccaag cgaaacatcg catcgagcga gcacgtactc ggatggaagc
     7321 cggtcttgtc gatcaggatg atctggacga agagcatcag gggctcgcgc cagccgaact
     7381 gttcgccagg ctcaaggcgc gcatgcccga cggcgaggat ctcgtcgtga cccatggcga
     7441 tgcctgcttg ccgaatatca tggtggaaaa tggccgcttt tctggattca tcgactgtgg
     7501 ccggctgggt gtggcggacc gctatcagga catagcgttg gctacccgtg atattgctga
     7561 agagcttggc ggcgaatggg ctgaccgctt cctcgtgctt tacggtatcg ccgctcccga
     7621 ttcgcagcgc atcgccttct atcgccttct tgacgagttc ttctgaacgc gttaagtcga
     7681 caatcaacct ctggattaca aaatttgtga aagattgact ggtattctta actatgttgc
     7741 tccttttacg ctatgtggat acgctgcttt aatgcctttg tatcatgcta ttgcttcccg
     7801 tatggctttc attttctcct ccttgtataa atcctggttg ctgtctcttt atgaggagtt
     7861 gtggcccgtt gtcaggcaac gtggcgtggt gtgcactgtg tttgctgacg caacccccac
     7921 tggttggggc attgccacca cctgtcagct cctttccggg actttcgctt tccccctccc
     7981 tattgccacg gcggaactca tcgccgcctg ccttgcccgc tgctggacag gggctcggct
     8041 gttgggcact gacaattccg tggtgttgtc ggggaaatca tcgtcctttc cttggctgct
     8101 cgcctgtgtt gccacctgga ttctgcgcgg gacgtccttc tgctacgtcc cttcggccct
     8161 caatccagcg gaccttcctt cccgcggcct gctgccggct ctgcggcctc ttccgcgtct
     8221 tcgccttcgc cctcagacga gtcggatctc cctttgggcc gcctccccgc gtcgacttta
     8281 agaccaatga cttacaaggc agctgtagat cttagccact ttttaaaaga aaagggggga
     8341 ctggaagggc taattcactc ccaacgaaga caagatctgc tttttgcttg tactgggtct
     8401 ctctggttag accagatctg agcctgggag ctctctggct aactagggaa cccactgctt
     8461 aagcctcaat aaagcttgcc ttgagtgctt caagtagtgt gtgcccgtct gttgtgtgac
     8521 tctggtaact agagatccct cagacccttt tagtcagtgt ggaaaatctc tagcagggcc
     8581 cgtttaaacc cgctgatcag cctcgactgt gccttctagt tgccagccat ctgttgtttg
     8641 cccctccccc gtgccttcct tgaccctgga aggtgccact cccactgtcc tttcctaata
     8701 aaatgaggaa attgcatcgc attgtctgag taggtgtcat tctattctgg ggggtggggt
     8761 ggggcaggac agcaaggggg aggattggga agacaatagc aggcatgctg gggatgcggt
     8821 gggctctatg gcttctgagg cggaaagaac cagctggggc tctagggggt atccccacgc
     8881 gccctgtagc ggcgcattaa gcgcggcggg tgtggtggtt acgcgcagcg tgaccgctac
     8941 acttgccagc gccctagcgc ccgctccttt cgctttcttc ccttcctttc tcgccacgtt
     9001 cgccggcttt ccccgtcaag ctctaaatcg ggggctccct ttagggttcc gatttagtgc
     9061 tttacggcac ctcgacccca aaaaacttga ttagggtgat ggttcacgta gtgggccatc
     9121 gccctgatag acggtttttc gccctttgac gttggagtcc acgttcttta atagtggact
     9181 cttgttccaa actggaacaa cactcaaccc tatctcggtc tattcttttg atttataagg
     9241 gattttgccg atttcggcct attggttaaa aaatgagctg atttaacaaa aatttaacgc
     9301 gaattaattc tgtggaatgt gtgtcagtta gggtgtggaa agtccccagg ctccccagca
     9361 ggcagaagta tgcaaagcat gcatctcaat tagtcagcaa ccaggtgtgg aaagtcccca
     9421 ggctccccag caggcagaag tatgcaaagc atgcatctca attagtcagc aaccatagtc
     9481 ccgcccctaa ctccgcccat cccgccccta actccgccca gttccgccca ttctccgccc
     9541 catggctgac taattttttt tatttatgca gaggccgagg ccgcctctgc ctctgagcta
     9601 ttccagaagt agtgaggagg cttttttgga ggcctaggct tttgcaaaaa gctcccggga
     9661 gcttgtatat ccattttcgg atctgatcag cacgtgttga caattaatca tcggcatagt
     9721 atatcggcat agtataatac gacaaggtga ggaactaaac catggccaag ttgaccagtg
     9781 ccgttccggt gctcaccgcg cgcgacgtcg ccggagcggt cgagttctgg accgaccggc
     9841 tcgggttctc ccgggacttc gtggaggacg acttcgccgg tgtggtccgg gacgacgtga
     9901 ccctgttcat cagcgcggtc caggaccagg tggtgccgga caacaccctg gcctgggtgt
     9961 gggtgcgcgg cctggacgag ctgtacgccg agtggtcgga ggtcgtgtcc acgaacttcc
    10021 gggacgcctc cgggccggcc atgaccgaga tcggcgagca gccgtggggg cgggagttcg
    10081 ccctgcgcga cccggccggc aactgcgtgc acttcgtggc cgaggagcag gactgacacg
    10141 tgctacgaga tttcgattcc accgccgcct tctatgaaag gttgggcttc ggaatcgttt
    10201 tccgggacgc cggctggatg atcctccagc gcggggatct catgctggag ttcttcgccc
    10261 accccaactt gtttattgca gcttataatg gttacaaata aagcaatagc atcacaaatt
    10321 tcacaaataa agcatttttt tcactgcatt ctagttgtgg tttgtccaaa ctcatcaatg
    10381 tatcttatca tgtctgtata ccgtcgacct ctagctagag cttggcgtaa tcatggtcat
    10441 agctgtttcc tgtgtgaaat tgttatccgc tcacaattcc acacaacata cgagccggaa
    10501 gcataaagtg taaagcctgg ggtgcctaat gagtgagcta actcacatta attgcgttgc
    10561 gctcactgcc cgctttccag tcgggaaacc tgtcgtgcca gctgcattaa tgaatcggcc
    10621 aacgcgcggg gagaggcggt ttgcgtattg ggcgctcttc cgcttcctcg ctcactgact
    10681 cgctgcgctc ggtcgttcgg ctgcggcgag cggtatcagc tcactcaaag gcggtaatac
    10741 ggttatccac agaatcaggg gataacgcag gaaagaacat gtgagcaaaa ggccagcaaa
    10801 aggccaggaa ccgtaaaaag gccgcgttgc tggcgttttt ccataggctc cgcccccctg
    10861 acgagcatca caaaaatcga cgctcaagtc agaggtggcg aaacccgaca ggactataaa
    10921 gataccaggc gtttccccct ggaagctccc tcgtgcgctc tcctgttccg accctgccgc
    10981 ttaccggata cctgtccgcc tttctccctt cgggaagcgt ggcgctttct catagctcac
    11041 gctgtaggta tctcagttcg gtgtaggtcg ttcgctccaa gctgggctgt gtgcacgaac
    11101 cccccgttca gcccgaccgc tgcgccttat ccggtaacta tcgtcttgag tccaacccgg
    11161 taagacacga cttatcgcca ctggcagcag ccactggtaa caggattagc agagcgaggt
    11221 atgtaggcgg tgctacagag ttcttgaagt ggtggcctaa ctacggctac actagaagaa
    11281 cagtatttgg tatctgcgct ctgctgaagc cagttacctt cggaaaaaga gttggtagct
    11341 cttgatccgg caaacaaacc accgctggta gcggtggttt ttttgtttgc aagcagcaga
    11401 ttacgcgcag aaaaaaagga tctcaagaag atcctttgat cttttctacg gggtctgacg
    11461 ctcagtggaa cgaaaactca cgttaaggga ttttggtcat gagattatca aaaaggatct
    11521 tcacctagat ccttttaaat taaaaatgaa gttttaaatc aatctaaagt atatatgagt
    11581 aaacttggtc tgacagttac caatgcttaa tcagtgaggc acctatctca gcgatctgtc
    11641 tatttcgttc atccatagtt gcctgactcc ccgtcgtgta gataactacg atacgggagg
    11701 gcttaccatc tggccccagt gctgcaatga taccgcgaga cccacgctca ccggctccag
    11761 atttatcagc aataaaccag ccagccggaa gggccgagcg cagaagtggt cctgcaactt
    11821 tatccgcctc catccagtct attaattgtt gccgggaagc tagagtaagt agttcgccag
    11881 ttaatagttt gcgcaacgtt gttgccattg ctacaggcat cgtggtgtca cgctcgtcgt
    11941 ttggtatggc ttcattcagc tccggttccc aacgatcaag gcgagttaca tgatccccca
    12001 tgttgtgcaa aaaagcggtt agctccttcg gtcctccgat cgttgtcaga agtaagttgg
    12061 ccgcagtgtt atcactcatg gttatggcag cactgcataa ttctcttact gtcatgccat
    12121 ccgtaagatg cttttctgtg actggtgagt actcaaccaa gtcattctga gaatagtgta
    12181 tgcggcgacc gagttgctct tgcccggcgt caatacggga taataccgcg ccacatagca
    12241 gaactttaaa agtgctcatc attggaaaac gttcttcggg gcgaaaactc tcaaggatct
    12301 taccgctgtt gagatccagt tcgatgtaac ccactcgtgc acccaactga tcttcagcat
    12361 cttttacttt caccagcgtt tctgggtgag caaaaacagg aaggcaaaat gccgcaaaaa
    12421 agggaataag ggcgacacgg aaatgttgaa tactcatact cttccttttt caatattatt
    12481 gaagcattta tcagggttat tgtctcatga gcggatacat atttgaatgt atttagaaaa
    12541 ataaacaaat aggggttccg cgcacatttc cccgaaaagt gccacctgac gtcgacggat
    12601 cgggagatct cccgatcccc tatggtgcac tctcagtaca atctgctctg atgccgcata
    12661 gttaagccag tatctgctcc ctgcttgtgt gttggaggtc gctgagtagt gcgcgagcaa
    12721 aatttaagct acaacaaggc aaggcttgac cgacaattgc atgaagaatc tgcttagggt
    12781 taggcgtttt gcgctgcttc gcgatgtacg ggccagatat acgcgcgcgt tgacattgat
    12841 tattgactag ttattaatag taatcaatta cggggtcatt agttcatagc ccatatatgg
    12901 agttccgcgt tacataactt acggtaaatg gcccgcctgg ctgaccgccc aacgaccccc
    12961 gcccattgac gtcaataatg acgtatgttc ccatagtaac gccaataggg actttccatt
    13021 gacgtcaatg ggtggagtat ttacggtaaa ctgcccactt ggcagtacat caagtgtatc
    13081 atatgccaag tacgccccct attgacgtca atgacggtaa atggcccgcc tggcattatg
    13141 cccagtacat gaccttatgg gactttccta cttggcagta catctacgta ttagtcatcg
    13201 ctattaccat gg
//
