LOCUS       sequence-463051-       13136 bp    DNA     circular SYN 15-DEC-2025
DEFINITION  Human ETFDH Gene Knockout (gRNA 1).
ACCESSION   .
VERSION     .
KEYWORDS    .
SOURCE      synthetic DNA construct
  ORGANISM  synthetic DNA construct
REFERENCE   1  (bases 1 to 13136)
  AUTHORS   Papadopoli D, Palia R, Jovanovic P, Tabaries S, Ciccolini E, 
            Sabourin V, Igelmann S, McLaughlan S, Zhan L, Kim H, Chekkal N, 
            Szkop KJ, Bertomeu T, Zeng J, Vassalakis J, Afzali F, Mzoughi S, 
            Guccione E, Tyers M, Avizonis D, Larsson O, Postovit LM, Djuranovic 
            S, Ursini-Siegel J, Siegel PM, Pollak M, Topisirovic I
  TITLE     Mitochondrial ETF insufficiency drives neoplastic growth by 
            selectively optimizing cancer bioenergetics
  JOURNAL   bioRxiv 2024.10.25.620155
REFERENCE   2  (bases 1 to 13136)
  AUTHORS   .
  TITLE     Direct Submission
  JOURNAL   Exported Dec 15, 2025 from SnapGene Server 8.0.1
            https://www.snapgene.com
COMMENT     SGRef: number: 1; type: "Journal Article"; journalName: "bioRxiv 
            2024.10.25.620155"
COMMENT     Sequence Label: hETFDH CRISPR_1
FEATURES             Location/Qualifiers
     source          1..13136
                     /mol_type="other DNA"
                     /organism="synthetic DNA construct"
     promoter        complement(41..370)
                     /label=SV40 promoter
                     /note="SV40 enhancer and early promoter"
     rep_origin      55..190
                     /label=SV40 ori
                     /note="SV40 origin of replication"
     primer_bind     complement(109..128)
                     /label=SV40pro-F
                     /note="SV40 promoter/origin, forward primer"
     primer_bind     355..375
                     /label=pBABE 3'
                     /note="SV40 enhancer, reverse primer for pBABE vectors"
     rep_origin      complement(384..812)
                     /direction=LEFT
                     /label=f1 ori
                     /note="f1 bacteriophage origin of replication; arrow 
                     indicates direction of (+) strand synthesis"
     primer_bind     complement(494..515)
                     /label=F1ori-F
                     /note="F1 origin, forward primer"
     primer_bind     706..725
                     /label=F1ori-R
                     /note="F1 origin, reverse primer"
     polyA_signal    858..1082
                     /label=bGH poly(A) signal
                     /note="bovine growth hormone polyadenylation signal"
     primer_bind     1071..1088
                     /label=BGH-rev
                     /note="Bovine growth hormone terminator, reverse primer. 
                     Also called BGH reverse"
     LTR             1114..1347
                     /label=3' LTR (Delta-U3)
                     /note="self-inactivating 3' long terminal repeat (LTR) from
                     HIV-1"
     misc_feature    1419..2007
                     /label=WPRE
                     /note="woodchuck hepatitis virus posttranscriptional 
                     regulatory element"
     CDS             1525..1536
                     /codon_start=1
                     /product="Factor Xa recognition and cleavage site"
                     /label=Factor Xa site
                     /translation="IEGR"
     primer_bind     1934..1954
                     /label=WPRE-R
                     /note="WPRE, reverse primer"
     CDS             complement(join(2026..2736,2737..2739,2740..2742))
                     /codon_start=1
                     /product="enhanced GFP"
                     /label=EGFP
                     /translation="MVSKGEELFTGVVPILVELDGDVNGHKFSVSGEGEGDATYGKLTL
                     KFICTTGKLPVPWPTLVTTLTYGVQCFSRYPDHMKQHDFFKSAMPEGYVQERTIFFKDD
                     GNYKTRAEVKFEGDTLVNRIELKGIDFKEDGNILGHKLEYNYNSHNVYIMADKQKNGIK
                     VNFKIRHNIEDGSVQLADHYQQNTPIGDGPVLLPDNHYLSTQSALSKDPNEKRDHMVLL
                     EFVTAAGITLGMDELYK"
     primer_bind     complement(2068..2089)
                     /label=EGFP-C
                     /note="EGFP, forward primer"
     primer_bind     2417..2436
                     /label=EXFP-R
                     /note="For distinguishing EGFP variants, reverse primer"
     CDS             complement(2743..2799)
                     /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             complement(2809..2832)
                     /codon_start=1
                     /product="FLAG(R) epitope tag, followed by an enterokinase 
                     cleavage site"
                     /label=FLAG
                     /translation="DYKDDDDK"
     CDS             complement(2833..2880)
                     /codon_start=1
                     /product="bipartite nuclear localization signal from 
                     nucleoplasmin"
                     /label=nucleoplasmin NLS
                     /translation="KRPAATKKAGQAKKKK"
     CDS             complement(2881..6984)
                     /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             complement(2881..6984)
                     /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"
     regulatory      6981..6990
                     /label=Kozak sequence
                     /note="vertebrate consensus sequence for strong initiation 
                     of translation (Kozak, 1987)"
                     /regulatory_class="other"
     promoter        complement(7009..7220)
                     /label=EF-1-alpha core promoter
                     /note="core promoter for human elongation factor 
                     EF-1-alpha"
     misc_RNA        complement(7282..7357)
                     /label=gRNA scaffold
                     /note="guide RNA scaffold for the Streptococcus pyogenes 
                     CRISPR/Cas9 system"
     promoter        complement(7379..7627)
                     /label=U6 promoter
                     /note="RNA polymerase III promoter for human U6 snRNA 
                     (Domitrovich & Kunkel, 2003)"
     primer_bind     complement(7437..7456)
                     /label=LKO.1 5'
                     /note="Human U6 promoter, forward primer"
     primer_bind     complement(7607..7627)
                     /label=hU6-F
                     /note="Human U6 promoter, forward primer"
     misc_feature    7678..7795
                     /label=cPPT/CTS
                     /note="central polypurine tract and central termination 
                     sequence of HIV-1"
     CDS             complement(8093..8137)
                     /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    8322..8555
                     /label=RRE
                     /note="The Rev response element (RRE) of HIV-1 allows for 
                     Rev-dependent mRNA export from the nucleus to the 
                     cytoplasm."
     misc_feature    9048..9173
                     /label=HIV-1 Psi
                     /note="packaging signal of human immunodeficiency virus 
                     type 1"
     LTR             9220..9400
                     /label=5' LTR (truncated)
                     /note="truncated 5' long terminal repeat (LTR) from HIV-1"
     promoter        complement(9418..9616)
                     /label=CMV promoter
                     /note="human cytomegalovirus (CMV) immediate early 
                     promoter"
     primer_bind     complement(9443..9463)
                     /label=CMV-F
                     /note="Human CMV immediate early promoter, forward primer"
     enhancer        9618..9997
                     /label=CMV enhancer
                     /note="human cytomegalovirus immediate early enhancer"
     primer_bind     10169..10188
                     /label=pRS-marker
                     /note="pRS vectors, use to sequence yeast selectable 
                     marker"
     promoter        10263..10367
                     /gene="bla"
                     /label=AmpR promoter
     CDS             join(10368..10436,10437..11228)
                     /codon_start=1
                     /gene="bla"
                     /product="beta-lactamase"
                     /label=AmpR
                     /note="confers resistance to ampicillin, carbenicillin, and
                     related antibiotics"
                     /translation="MSIQHFRVALIPFFAAFCLPVFAHPETLVKVKDAEDQLGARVGYI
                     ELDLNSGKILESFRPEERFPMMSTFKVLLCGAVLSRIDAGQEQLGRRIHYSQNDLVEYS
                     PVTEKHLTDGMTVRELCSAAITMSDNTAANLLLTTIGGPKELTAFLHNMGDHVTRLDRW
                     EPELNEAIPNDERDTTMPVAMATTLRKLLTGELLTLASRQQLIDWMEADKVAGPLLRSA
                     LPAGWFIADKSGAGERGSRGIIAALGPDGKPSRIVVIYTTGSQATMDERNRQIAEIGAS
                     LIKHW"
     primer_bind     complement(10586..10605)
                     /label=Amp-R
                     /note="Ampicillin resistance gene, reverse primer"
     rep_origin      11399..11987
                     /direction=RIGHT
                     /label=ori
                     /note="high-copy-number ColE1/pMB1/pBR322/pUC origin of 
                     replication"
     primer_bind     11888..11907
                     /label=pBR322ori-F
                     /note="pBR322 origin, forward primer"
     primer_bind     12141..12158
                     /label=L4440
                     /note="L4440 vector, forward primer"
     protein_bind    12275..12296
                     /label=CAP binding site
                     /bound_moiety="E. coli catabolite activator protein"
                     /note="CAP binding activates transcription in the presence 
                     of cAMP."
     promoter        join(12311..12316,12317..12334,12335..12341)
                     /label=lac promoter
                     /note="promoter for the E. coli lac operon"
     protein_bind    12349..12365
                     /label=lac operator
                     /bound_moiety="lac repressor encoded by lacI"
                     /note="The lac repressor binds to the lac operator to 
                     inhibit transcription in E. coli. This inhibition can be 
                     relieved by adding lactose or 
                     isopropyl-beta-D-thiogalactopyranoside (IPTG)."
     primer_bind     12354..12376
                     /label=M13/pUC Reverse
                     /note="In lacZ gene"
     primer_bind     12373..12389
                     /label=M13 rev
                     /note="common sequencing primer, one of multiple similar 
                     variants"
     primer_bind     12373..12389
                     /label=M13 Reverse
                     /note="In lacZ gene. Also called M13-rev"
     polyA_signal    12438..12559
                     /label=SV40 poly(A) signal
                     /note="SV40 polyadenylation signal"
     primer_bind     complement(12449..12468)
                     /label=EBV-rev
                     /note="SV40 polyA terminator, reverse primer"
     primer_bind     12503..12522
                     /label=SV40pA-R
                     /note="SV40 polyA, reverse primer"
     CDS             complement(12689..13063)
                     /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"
     promoter        complement(13082..13129)
                     /label=EM7 promoter
                     /note="synthetic bacterial promoter "
ORIGIN
        1 gatcagatcc gaaaatggat atacaagctc ccgggagctt tttgcaaaag cctaggcctc
       61 caaaaaagcc tcctcactac ttctggaata gctcagaggc agaggcggcc tcggcctctg
      121 cataaataaa aaaaattagt cagccatggg gcggagaatg ggcggaactg ggcggagtta
      181 ggggcgggat gggcggagtt aggggcggga ctatggttgc tgactaattg agatgcatgc
      241 tttgcatact tctgcctgct ggggagcctg gggactttcc acacctggtt gctgactaat
      301 tgagatgcat gctttgcata cttctgcctg ctggggagcc tggggacttt ccacacccta
      361 actgacacac attccacaga attaattcgc gttaaatttt tgttaaatca gctcattttt
      421 taaccaatag gccgaaatcg gcaaaatccc ttataaatca aaagaataga ccgagatagg
      481 gttgagtgtt gttccagttt ggaacaagag tccactatta aagaacgtgg actccaacgt
      541 caaagggcga aaaaccgtct atcagggcga tggcccacta cgtgaaccat caccctaatc
      601 aagttttttg gggtcgaggt gccgtaaagc actaaatcgg aaccctaaag ggagcccccg
      661 atttagagct tgacggggaa agccggcgaa cgtggcgaga aaggaaggga agaaagcgaa
      721 aggagcgggc gctagggcgc tggcaagtgt agcggtcacg ctgcgcgtaa ccaccacacc
      781 cgccgcgctt aatgcgccgc tacagggcgc gtggggatac cccctagagc cccagctggt
      841 tctttccgcc tcagaagcca tagagcccac cgcatcccca gcatgcctgc tattgtcttc
      901 ccaatcctcc cccttgctgt cctgccccac cccacccccc agaatagaat gacacctact
      961 cagacaatgc gatgcaattt cctcatttta ttaggaaagg acagtgggag tggcaccttc
     1021 cagggtcaag gaaggcacgg gggaggggca aacaacagat ggctggcaac tagaaggcac
     1081 agtcgaggct gatcagcggg tttaaacggg ccctgctaga gattttccac actgactaaa
     1141 agggtctgag ggatctctag ttaccagagt cacacaacag acgggcacac actacttgaa
     1201 gcactcaagg caagctttat tgaggcttaa gcagtgggtt ccctagttag ccagagagct
     1261 cccaggctca gatctggtct aaccagagag acccagtaca agcaaaaagc agatcttgtc
     1321 ttcgttggga gtgaattagc ccttccagtc cccccttttc ttttaaaaag tggctaagat
     1381 ctacagctgc cttgtaagtc attggtctta aagtcgacgc ggggaggcgg cccaaaggga
     1441 gatccgactc gtctgagggc gaaggcgaag acgcggaaga ggccgcagag ccggcagcag
     1501 gccgcgggaa ggaaggtccg ctggattgag ggccgaaggg acgtagcaga aggacgtccc
     1561 gcgcagaatc caggtggcaa cacaggcgag cagccaagga aaggacgatg atttccccga
     1621 caacaccacg gaattgtcag tgcccaacag ccgagcccct gtccagcagc gggcaaggca
     1681 ggcggcgatg agttccgccg tggcaatagg gagggggaaa gcgaaagtcc cggaaaggag
     1741 ctgacaggtg gtggcaatgc cccaaccagt gggggttgcg tcagcaaaca cagtgcacac
     1801 cacgccacgt tgcctgacaa cgggccacaa ctcctcataa agagacagca accaggattt
     1861 atacaaggag gagaaaatga aagccatacg ggaagcaata gcatgataca aaggcattaa
     1921 agcagcgtat ccacatagcg taaaaggagc aacatagtta agaataccag tcaatctttc
     1981 acaaattttg taatccagag gttgattgtc gacttaacgc gtttacttgt acagctcgtc
     2041 catgccgaga gtgatcccgg cggcggtcac gaactccagc aggaccatgt gatcgcgctt
     2101 ctcgttgggg tctttgctca gggcggactg ggtgctcagg tagtggttgt cgggcagcag
     2161 cacggggccg tcgccgatgg gggtgttctg ctggtagtgg tcggcgagct gcacgctgcc
     2221 gtcctcgatg ttgtggcgga tcttgaagtt caccttgatg ccgttcttct gcttgtcggc
     2281 catgatatag acgttgtggc tgttgtagtt gtactccagc ttgtgcccca ggatgttgcc
     2341 gtcctccttg aagtcgatgc ccttcagctc gatgcggttc accagggtgt cgccctcgaa
     2401 cttcacctcg gcgcgggtct tgtagttgcc gtcgtccttg aagaagatgg tgcgctcctg
     2461 gacgtagcct tcgggcatgg cggacttgaa gaagtcgtgc tgcttcatgt ggtcggggta
     2521 gcggctgaag cactgcacgc cgtaggtcag ggtggtcacg agggtgggcc agggcacggg
     2581 cagcttgccg gtggtgcaga tgaacttcag ggtcagcttg ccgtaggtgg catcgccctc
     2641 gccctcgccg gacacgctga acttgtggcc gtttacgtcg ccgtccagct cgaccaggat
     2701 gggcaccacc ccggtgaaca gctcttcgcc cttagacacc atcggtccag gattctcttc
     2761 gacatctccg gcttgtttca gcagagagaa gtttgttgcg ccggatccct tatcgtcatc
     2821 gtctttgtaa tctttcttct tcttagcctg tccagccttc tttgtggcgg caggtcgctt
     2881 gtcgcctccc agctgagaca ggtcgatccg tgtctcgtac aggccggtga tgctctggtg
     2941 gatcagggtg gcgtccagca cctctttggt gctggtgtac ctcttccggt cgatggtggt
     3001 gtcaaagtac ttgaaggcgg caggggctcc cagattggtc agggtaaaca ggtggatgat
     3061 attctcggcc tgctctctga tgggcttatc ccggtgcttg ttgtaggcgg acagcacttt
     3121 gtccagatta gcgtcggcca ggatcactct cttggagaac tcgctgatct gctcgatgat
     3181 ctcgtccagg tagtgcttgt gctgttccac aaacagctgt ttctgctcat tatcctcggg
     3241 ggagcccttc agcttctcat agtggctggc caggtacagg aagttcacat atttggaggg
     3301 cagggccagt tcgtttccct tctgcagttc gccggcagag gccagcattc tcttccggcc
     3361 gttttccagc tcgaacaggg agtacttagg cagcttgatg atcaggtcct ttttcacttc
     3421 tttgtagccc ttggcttcca gaaagtcgat gggattcttc tcgaagctgc ttctttccat
     3481 gatggtgatc cccagcagct ctttcacact cttcagtttc ttggacttgc ccttttccac
     3541 tttggccacc accagcacag aataggccac ggtggggctg tcgaagccgc cgtacttctt
     3601 agggtcccag tccttctttc tggcgatcag cttatcgctg ttcctcttgg gcaggataga
     3661 ctctttgctg aagccgcctg tctgcacctc ggtctttttc acgatattca cttggggcat
     3721 gctcagcact ttccgcacgg tggcaaaatc ccggccctta tcccacacga tctccccggt
     3781 ttcgccgttt gtctcgatca gaggccgctt ccggatctcg ccgttggcca gggtaatctc
     3841 ggtcttgaaa aagttcatga tgttgctgta gaagaagtac ttggcggtag ccttgccgat
     3901 ttcctgctcg ctcttggcga tcatcttccg cacgtcgtac accttgtagt cgccgtacac
     3961 gaactcgctt tccagcttag ggtacttttt gatcagggcg gttcccacga cggcgttcag
     4021 gtaggcgtcg tgggcgtggt ggtagttgtt gatctcgcgc actttgtaaa actggaaatc
     4081 cttccggaaa tcggacacca gcttggactt cagggtgatc actttcactt cccggatcag
     4141 cttgtcattc tcgtcgtact tagtgttcat ccgggagtcc aggatctgtg ccacgtgctt
     4201 tgtgatctgc cgggtttcca ccagctgtct cttgatgaag ccggccttat ccagttcgct
     4261 caggccgcct ctctcggcct tggtcagatt gtcgaacttt ctctgggtaa tcagcttggc
     4321 gttcagcagc tgccgccagt agttcttcat cttcttcacg acctcttcgg agggcacgtt
     4381 gtcgctcttg ccccggttct tgtcgcttct ggtcagcacc ttgttgtcga tggagtcgtc
     4441 cttcagaaag ctctgaggca cgatatggtc cacatcgtag tcggacagcc ggttgatgtc
     4501 cagttcctgg tccacgtaca tatcccgccc attctgcagg tagtacaggt acagcttctc
     4561 gttctgcagc tgggtgtttt ccacggggtg ttctttcagg atctggctgc ccagctcttt
     4621 gatgccctct tcgatccgct tcattctctc gcggctgttc ttctgtccct tctgggtggt
     4681 ctggttctct ctggccattt cgatcacgat gttctcgggc ttgtgccggc ccatcacttt
     4741 cacgagctcg tccaccacct tcactgtctg caggatgccc ttcttaatgg cggggctgcc
     4801 ggccagattg gcaatgtgct cgtgcaggct atcgccctgg ccggacacct gggctttctg
     4861 gatgtcctct ttaaaggtca ggctgtcgtc gtggatcagc tgcatgaagt ttctgttggc
     4921 gaagccgtcg gacttcagga aatccaggat tgtcttgccg gactgcttgt cccggatgcc
     4981 gttgatcagc ttccggctca gcctgcccca gccggtgtat ctccgccgct tcagctgctt
     5041 catcactttg tcgtcgaaca ggtgggcata ggttttcagc cgttcctcga tcatctctct
     5101 gtcctcaaac agtgtcaggg tcagcacgat atcttccaga atgtcctcgt tttcctcatt
     5161 gtccaggaag tccttgtcct tgataatttt cagcagatcg tggtatgtgc ccagggaggc
     5221 gttgaaccga tcttccacgc cggagatttc cacggagtcg aagcactcga ttttcttgaa
     5281 gtagtcctct ttcagctgct tcacggtcac tttccggttg gtcttgaaca gcaggtccac
     5341 gatggccttt ttctgctcgc cgctcaggaa ggcgggcttt ctcattccct cggtcacgta
     5401 tttcactttg gtcagctcgt tatacacggt gaagtactcg tacagcaggc tgtgcttggg
     5461 cagcaccttc tcgttgggca ggttcttatc gaagttggtc atccgctcga tgaagctctg
     5521 ggcggaagcg cccttgtcca ccacttcctc gaagttccag ggggtgatgg tttcctcgct
     5581 ctttctggtc atccaggcga atctgctgtt tcccctggcc agagggccca cgtagtaggg
     5641 gatgcggaag gtcaggatct tctcgatctt ttcccggttg tccttcagga atgggtaaaa
     5701 atcttcctgc cgccgcagaa tggcgtgcag ctctcccagg tggatctggt gggggatgct
     5761 gccgttgtcg aaggtccgct gcttccgcag caggtcctct ctgttcagct tcacgagcag
     5821 ttcctcggtg ccgtccatct tttccaggat gggcttgatg aacttgtaga actcttcctg
     5881 gctggctccg ccgtcaatgt agccggcgta gccgttcttg ctctggtcga agaaaatctc
     5941 tttgtacttc tcaggcagct gctgccgcac gagagctttc agcagggtca ggtcctggtg
     6001 gtgctcgtcg tatctcttga tcatagaggc gctcaggggg gccttggtga tctcggtgtt
     6061 cactctcagg atgtcgctca gcaggatggc gtcggacagg ttcttggcgg ccagaaacag
     6121 gtcggcgtac tggtcgccga tctgggccag caggttgtcc aggtcgtcgt cgtaggtgtc
     6181 cttgctcagc tgcagtttgg catcctcggc caggtcgaag ttgctcttga agttgggggt
     6241 caggcccagg ctcagggcaa tcaggtttcc gaacaggcca ttcttcttct cgccgggcag
     6301 ctgggcgatc agattttcca gccgtctgct cttgctcagt ctggcagaca ggatggcctt
     6361 ggcgtccacg ccgctggcgt tgatggggtt ttcctcgaac agctggttgt aggtctgcac
     6421 cagctggatg aacagcttgt ccacgtcgct gttgtcgggg ttcaggtcgc cctcgatcag
     6481 gaagtggccc cggaacttga tcatgtgggc cagggccaga tagatcagcc gcaggtcggc
     6541 cttgtcggtg ctgtccacca gtttctttct caggtggtag atggtggggt acttctcgtg
     6601 gtaggccacc tcgtccacga tgttgccgaa gatggggtgc cgctcgtgct tcttatcctc
     6661 ttccaccagg aaggactctt ccagtctgtg gaagaagctg tcgtccacct tggccatctc
     6721 gttgctgaag atctcttgca gatagcagat ccggttcttc cgtctggtgt atcttcttct
     6781 ggcggttctc ttcagccggg tggcctcggc tgtttcgccg ctgtcgaaca gcagggctcc
     6841 gatcaggttc ttcttgatgc tgtgccggtc ggtgttgccc agcaccttga atttcttgct
     6901 gggcaccttg tactcgtcgg tgatcacggc ccagcccaca gagttggtgc cgatgtccag
     6961 gccgatgctg tacttcttgt ccatggtggc agcgctctag aaccggtcct gtgttctggc
     7021 ggcaaacccg ttgcgaaaaa gaacgttcac ggcgactact gcacttatat acggttctcc
     7081 cccaccctcg ggaaaaaggc ggagccagta cacgacatca ctttcccagt ttaccccgcg
     7141 ccaccttctc taggcaccgg atcaattgcc gacccctccc cccaacttct cggggactgt
     7201 gggcgatgtg cgctctgccc actgacgggc accggagcca attcccactc ctttcaagac
     7261 ctagctagcg aattcaaaaa agcaccgact cggtgccact ttttcaagtt gataacggac
     7321 tagccttatt ttaacttgct atttctagct ctaaaactcc tagcattcgg ccaacctcgg
     7381 tgtttcgtcc tttccacaag atatataaag ccaagaaatc gaaatacttt caagttacgg
     7441 taagcatatg atagtccatt ttaaaacata attttaaaac tgcaaactac ccaagaaatt
     7501 attactttct acgtcacgta ttttgtacta atatctttgt gtttacagtc aaattaattc
     7561 taattatctc tctaacagcc ttgtatcgta tatgcaaata tgaaggaatc atgggaaata
     7621 ggccctcggt accttaatta accaaactgg atctctgctg tccctgtaat aaacccgaaa
     7681 attttgaatt tttgtaattt gtttttgtaa ttctttagtt tgtatgtctg ttgctattat
     7741 gtctactatt ctttcccctg cactgtaccc cccaatcccc ccttttcttt taaaattgtg
     7801 gatgaatact gccatttgtc tgcagaattg gcgcacgcag tgccgatccg ttcactaatc
     7861 gaatggatct gtctctgtct ctctctccac cttcttcttc tattccttcg ggcctgtcgg
     7921 gtcccctcgg ggttgggagg tgggtctgaa acgataatgg tgaatatccc tgcctaactc
     7981 tattcactat agaaagtaca gcaaaaacta ttcttaaacc taccaagcct cctactatca
     8041 ttatgaataa ttttatatac cacagccaat ttgttatgtt aaaccaattc cacaaacttg
     8101 cccatttatc taattccaat aattcttgtt cattcttttc ttgctggttt tgcgattctt
     8161 caattaagga gtgtattaag cttgtgtaat tgttaatttc tctgtcccac tccatccagg
     8221 tcgtgtgatt ccaaatctgt tccagagatt tattactcca actagcattc caaggcacag
     8281 cagtggtgca aatgagtttt ccagagcaac cccaaatccc caggagctgt tgatccttta
     8341 ggtatctttc cacagccagg attcttgcct ggagctgctt gatgccccag actgtgagtt
     8401 gcaacagatg ctgttgcgcc tcaatagccc tcagcaaatt gttctgctgc tgcactatac
     8461 cagacaataa ttgtctggcc tgtaccgtca gcgtcattga cgctgcgccc atagtgcttc
     8521 ctgctgctcc caagaaccca aggaacaaag ctcctattcc cactgctctt ttttctctct
     8581 gcaccactct tctctttgcc ttggtgggtg ctactcctaa tggttcaatt tttactactt
     8641 tatatttata taattcactt ctccaattgt ccctcatatc tcctcctcca ggtctgaaga
     8701 tcagcggccg cttgctgtgc ggtggtctta cttttgtttt gctcttcctc tatcttgtct
     8761 aaagcttcct tggtgtcttt tatctctatc ctttgatgca cacaatagag ggttgctact
     8821 gtattatata atgatctaag ttcttctgat cctgtctgaa gggatggttg tagctgtccc
     8881 agtatttgtc tacagccttc tgatgtttct aacaggccag gattaactgc gaatcgttct
     8941 agctccctgc ttgcccatac tatatgtttt aatttatatt ttttctttcc ccctggcctt
     9001 aaccgaattt tttcccatcg cgatctaatt ctcccccgct taatactgac gctctcgcac
     9061 ccatctctct ccttctagcc tccgctagtc aaaatttttg gcgtactcac cagtcgccgc
     9121 ccctcgcctc ttgccgtgcg cgcttcagca agccgagtcc tgcgtcgaga gagctcctct
     9181 ggtttccctt tcgctttcaa gtccctgttc gggcgccact gctagagatt ttccacactg
     9241 actaaaaggg tctgagggat ctctagttac cagagtcaca caacagacgg gcacacacta
     9301 cttgaagcac tcaaggcaag ctttattgag gcttaagcag tgggttccct agttagccag
     9361 agagctccca ggctcagatc tggtctaacc agagagaccc agtacaggca aaacgcgctg
     9421 cttatataga cctcccaccg tacacgccta ccgcccattt gcgtcaatgg ggcggagttg
     9481 ttacgacatt ttggaaagtc ccgttgattt tggtgccaaa acaaactccc attgacgtca
     9541 atggggtgga gacttggaaa tccccgtgag tcaaaccgct atccacgccc attgatgtac
     9601 tgccaaaacc gcatcaccat ggtaatagcg atgactaata cgtagatgta ctgccaagta
     9661 ggaaagtccc ataaggtcat gtactgggca taatgccagg cgggccattt accgtcattg
     9721 acgtcaatag ggggcgtact tggcatatga tacacttgat gtactgccaa gtgggcagtt
     9781 taccgtaaat actccaccca ttgacgtcaa tggaaagtcc ctattggcgt tactatggga
     9841 acatacgtca ttattgacgt caatgggcgg gggtcgttgg gcggtcagcc aggcgggcca
     9901 tttaccgtaa gttatgtaac gcggaactcc atatatgggc tatgaactaa tgaccccgta
     9961 attgattact attaataact agtcaataat caatgtcaac gcgtatatct ggcccgtaca
    10021 tcgcgaagca gcgcaaaacg cctaacccta agcagattct tcatgcaatt gtcggtcaag
    10081 ccttgccttg ttgtagctta aattttgctc gcgcactact cagcgacctc caacacacaa
    10141 gcagggagca gatactggct taactatgcg gcatcagagc agattgtact gagagtgcac
    10201 cataggggat cgggagatct cccgatccgt cgacgtcagg tggcactttt cggggaaatg
    10261 tgcgcggaac ccctatttgt ttatttttct aaatacattc aaatatgtat ccgctcatga
    10321 gacaataacc ctgataaatg cttcaataat attgaaaaag gaagagtatg agtattcaac
    10381 atttccgtgt cgcccttatt cccttttttg cggcattttg ccttcctgtt tttgctcacc
    10441 cagaaacgct ggtgaaagta aaagatgctg aagatcagtt gggtgcacga gtgggttaca
    10501 tcgaactgga tctcaacagc ggtaagatcc ttgagagttt tcgccccgaa gaacgttttc
    10561 caatgatgag cacttttaaa gttctgctat gtggcgcggt attatcccgt attgacgccg
    10621 ggcaagagca actcggtcgc cgcatacact attctcagaa tgacttggtt gagtactcac
    10681 cagtcacaga aaagcatctt acggatggca tgacagtaag agaattatgc agtgctgcca
    10741 taaccatgag tgataacact gcggccaact tacttctgac aacgatcgga ggaccgaagg
    10801 agctaaccgc ttttttgcac aacatggggg atcatgtaac tcgccttgat cgttgggaac
    10861 cggagctgaa tgaagccata ccaaacgacg agcgtgacac cacgatgcct gtagcaatgg
    10921 caacaacgtt gcgcaaacta ttaactggcg aactacttac tctagcttcc cggcaacaat
    10981 taatagactg gatggaggcg gataaagttg caggaccact tctgcgctcg gcccttccgg
    11041 ctggctggtt tattgctgat aaatctggag ccggtgagcg tgggtctcgc ggtatcattg
    11101 cagcactggg gccagatggt aagccctccc gtatcgtagt tatctacacg acggggagtc
    11161 aggcaactat ggatgaacga aatagacaga tcgctgagat aggtgcctca ctgattaagc
    11221 attggtaact gtcagaccaa gtttactcat atatacttta gattgattta aaacttcatt
    11281 tttaatttaa aaggatctag gtgaagatcc tttttgataa tctcatgacc aaaatccctt
    11341 aacgtgagtt ttcgttccac tgagcgtcag accccgtaga aaagatcaaa ggatcttctt
    11401 gagatccttt ttttctgcgc gtaatctgct gcttgcaaac aaaaaaacca ccgctaccag
    11461 cggtggtttg tttgccggat caagagctac caactctttt tccgaaggta actggcttca
    11521 gcagagcgca gataccaaat actgttcttc tagtgtagcc gtagttaggc caccacttca
    11581 agaactctgt agcaccgcct acatacctcg ctctgctaat cctgttacca gtggctgctg
    11641 ccagtggcga taagtcgtgt cttaccgggt tggactcaag acgatagtta ccggataagg
    11701 cgcagcggtc gggctgaacg gggggttcgt gcacacagcc cagcttggag cgaacgacct
    11761 acaccgaact gagataccta cagcgtgagc tatgagaaag cgccacgctt cccgaaggga
    11821 gaaaggcgga caggtatccg gtaagcggca gggtcggaac aggagagcgc acgagggagc
    11881 ttccaggggg aaacgcctgg tatctttata gtcctgtcgg gtttcgccac ctctgacttg
    11941 agcgtcgatt tttgtgatgc tcgtcagggg ggcggagcct atggaaaaac gccagcaacg
    12001 cggccttttt acggttcctg gccttttgct ggccttttgc tcacatgttc tttcctgcgt
    12061 tatcccctga ttctgtggat aaccgtatta ccgcctttga gtgagctgat accgctcgcc
    12121 gcagccgaac gaccgagcgc agcgagtcag tgagcgagga agcggaagag cgcccaatac
    12181 gcaaaccgcc tctccccgcg cgttggccga ttcattaatg cagctggcac gacaggtttc
    12241 ccgactggaa agcgggcagt gagcgcaacg caattaatgt gagttagctc actcattagg
    12301 caccccaggc tttacacttt atgcttccgg ctcgtatgtt gtgtggaatt gtgagcggat
    12361 aacaatttca cacaggaaac agctatgacc atgattacgc caagctctag ctagaggtcg
    12421 acggtataca gacatgataa gatacattga tgagtttgga caaaccacaa ctagaatgca
    12481 gtgaaaaaaa tgctttattt gtgaaatttg tgatgctatt gctttatttg taaccattat
    12541 aagctgcaat aaacaagttg gggtgggcga agaactccag catgagatcc ccgcgctgga
    12601 ggatcatcca gccggcgtcc cggaaaacga ttccgaagcc caacctttca tagaaggcgg
    12661 cggtggaatc gaaatctcgt agcacgtgtc agtcctgctc ctcggccacg aagtgcacgc
    12721 agttgccggc cgggtcgcgc agggcgaact cccgccccca cggctgctcg ccgatctcgg
    12781 tcatggccgg cccggaggcg tcccggaagt tcgtggacac gacctccgac cactcggcgt
    12841 acagctcgtc caggccgcgc acccacaccc aggccagggt gttgtccggc accacctggt
    12901 cctggaccgc gctgatgaac agggtcacgt cgtcccggac cacaccggcg aagtcgtcct
    12961 ccacgaagtc ccgggagaac ccgagccggt cggtccagaa ctcgaccgct ccggcgacgt
    13021 cgcgcgcggt gagcaccgga acggcactgg tcaacttggc catggtttag ttcctcacct
    13081 tgtcgtatta tactatgccg atatactatg ccgatgatta attgtcaaca cgtgct
//
