LOCUS       sequence-466601-       13135 bp    DNA     circular SYN 15-DEC-2025
DEFINITION  Human ETFDH Gene Knockout (gRNA 2).
ACCESSION   .
VERSION     .
KEYWORDS    .
SOURCE      synthetic DNA construct
  ORGANISM  synthetic DNA construct
REFERENCE   1  (bases 1 to 13135)
  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 13135)
  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_2
FEATURES             Location/Qualifiers
     source          1..13135
                     /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(7378..7626)
                     /label=U6 promoter
                     /note="RNA polymerase III promoter for human U6 snRNA 
                     (Domitrovich & Kunkel, 2003)"
     primer_bind     complement(7436..7455)
                     /label=LKO.1 5'
                     /note="Human U6 promoter, forward primer"
     primer_bind     complement(7606..7626)
                     /label=hU6-F
                     /note="Human U6 promoter, forward primer"
     misc_feature    7677..7794
                     /label=cPPT/CTS
                     /note="central polypurine tract and central termination 
                     sequence of HIV-1"
     CDS             complement(8092..8136)
                     /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    8321..8554
                     /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    9047..9172
                     /label=HIV-1 Psi
                     /note="packaging signal of human immunodeficiency virus 
                     type 1"
     LTR             9219..9399
                     /label=5' LTR (truncated)
                     /note="truncated 5' long terminal repeat (LTR) from HIV-1"
     promoter        complement(9417..9615)
                     /label=CMV promoter
                     /note="human cytomegalovirus (CMV) immediate early 
                     promoter"
     primer_bind     complement(9442..9462)
                     /label=CMV-F
                     /note="Human CMV immediate early promoter, forward primer"
     enhancer        9617..9996
                     /label=CMV enhancer
                     /note="human cytomegalovirus immediate early enhancer"
     primer_bind     10168..10187
                     /label=pRS-marker
                     /note="pRS vectors, use to sequence yeast selectable 
                     marker"
     promoter        10262..10366
                     /gene="bla"
                     /label=AmpR promoter
     CDS             join(10367..10435,10436..11227)
                     /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(10585..10604)
                     /label=Amp-R
                     /note="Ampicillin resistance gene, reverse primer"
     rep_origin      11398..11986
                     /direction=RIGHT
                     /label=ori
                     /note="high-copy-number ColE1/pMB1/pBR322/pUC origin of 
                     replication"
     primer_bind     11887..11906
                     /label=pBR322ori-F
                     /note="pBR322 origin, forward primer"
     primer_bind     12140..12157
                     /label=L4440
                     /note="L4440 vector, forward primer"
     protein_bind    12274..12295
                     /label=CAP binding site
                     /bound_moiety="E. coli catabolite activator protein"
                     /note="CAP binding activates transcription in the presence 
                     of cAMP."
     promoter        join(12310..12315,12316..12333,12334..12340)
                     /label=lac promoter
                     /note="promoter for the E. coli lac operon"
     protein_bind    12348..12364
                     /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     12353..12375
                     /label=M13/pUC Reverse
                     /note="In lacZ gene"
     primer_bind     12372..12388
                     /label=M13 rev
                     /note="common sequencing primer, one of multiple similar 
                     variants"
     primer_bind     12372..12388
                     /label=M13 Reverse
                     /note="In lacZ gene. Also called M13-rev"
     polyA_signal    12437..12558
                     /label=SV40 poly(A) signal
                     /note="SV40 polyadenylation signal"
     primer_bind     complement(12448..12467)
                     /label=EBV-rev
                     /note="SV40 polyA terminator, reverse primer"
     primer_bind     12502..12521
                     /label=SV40pA-R
                     /note="SV40 polyA, reverse primer"
     CDS             complement(12688..13062)
                     /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(13081..13128)
                     /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 ttttgtatcc ctacatcggt
     7381 gtttcgtcct ttccacaaga tatataaagc caagaaatcg aaatactttc aagttacggt
     7441 aagcatatga tagtccattt taaaacataa ttttaaaact gcaaactacc caagaaatta
     7501 ttactttcta cgtcacgtat tttgtactaa tatctttgtg tttacagtca aattaattct
     7561 aattatctct ctaacagcct tgtatcgtat atgcaaatat gaaggaatca tgggaaatag
     7621 gccctcggta ccttaattaa ccaaactgga tctctgctgt ccctgtaata aacccgaaaa
     7681 ttttgaattt ttgtaatttg tttttgtaat tctttagttt gtatgtctgt tgctattatg
     7741 tctactattc tttcccctgc actgtacccc ccaatccccc cttttctttt aaaattgtgg
     7801 atgaatactg ccatttgtct gcagaattgg cgcacgcagt gccgatccgt tcactaatcg
     7861 aatggatctg tctctgtctc tctctccacc ttcttcttct attccttcgg gcctgtcggg
     7921 tcccctcggg gttgggaggt gggtctgaaa cgataatggt gaatatccct gcctaactct
     7981 attcactata gaaagtacag caaaaactat tcttaaacct accaagcctc ctactatcat
     8041 tatgaataat tttatatacc acagccaatt tgttatgtta aaccaattcc acaaacttgc
     8101 ccatttatct aattccaata attcttgttc attcttttct tgctggtttt gcgattcttc
     8161 aattaaggag tgtattaagc ttgtgtaatt gttaatttct ctgtcccact ccatccaggt
     8221 cgtgtgattc caaatctgtt ccagagattt attactccaa ctagcattcc aaggcacagc
     8281 agtggtgcaa atgagttttc cagagcaacc ccaaatcccc aggagctgtt gatcctttag
     8341 gtatctttcc acagccagga ttcttgcctg gagctgcttg atgccccaga ctgtgagttg
     8401 caacagatgc tgttgcgcct caatagccct cagcaaattg ttctgctgct gcactatacc
     8461 agacaataat tgtctggcct gtaccgtcag cgtcattgac gctgcgccca tagtgcttcc
     8521 tgctgctccc aagaacccaa ggaacaaagc tcctattccc actgctcttt tttctctctg
     8581 caccactctt ctctttgcct tggtgggtgc tactcctaat ggttcaattt ttactacttt
     8641 atatttatat aattcacttc tccaattgtc cctcatatct cctcctccag gtctgaagat
     8701 cagcggccgc ttgctgtgcg gtggtcttac ttttgttttg ctcttcctct atcttgtcta
     8761 aagcttcctt ggtgtctttt atctctatcc tttgatgcac acaatagagg gttgctactg
     8821 tattatataa tgatctaagt tcttctgatc ctgtctgaag ggatggttgt agctgtccca
     8881 gtatttgtct acagccttct gatgtttcta acaggccagg attaactgcg aatcgttcta
     8941 gctccctgct tgcccatact atatgtttta atttatattt tttctttccc cctggcctta
     9001 accgaatttt ttcccatcgc gatctaattc tcccccgctt aatactgacg ctctcgcacc
     9061 catctctctc cttctagcct ccgctagtca aaatttttgg cgtactcacc agtcgccgcc
     9121 cctcgcctct tgccgtgcgc gcttcagcaa gccgagtcct gcgtcgagag agctcctctg
     9181 gtttcccttt cgctttcaag tccctgttcg ggcgccactg ctagagattt tccacactga
     9241 ctaaaagggt ctgagggatc tctagttacc agagtcacac aacagacggg cacacactac
     9301 ttgaagcact caaggcaagc tttattgagg cttaagcagt gggttcccta gttagccaga
     9361 gagctcccag gctcagatct ggtctaacca gagagaccca gtacaggcaa aacgcgctgc
     9421 ttatatagac ctcccaccgt acacgcctac cgcccatttg cgtcaatggg gcggagttgt
     9481 tacgacattt tggaaagtcc cgttgatttt ggtgccaaaa caaactccca ttgacgtcaa
     9541 tggggtggag acttggaaat ccccgtgagt caaaccgcta tccacgccca ttgatgtact
     9601 gccaaaaccg catcaccatg gtaatagcga tgactaatac gtagatgtac tgccaagtag
     9661 gaaagtccca taaggtcatg tactgggcat aatgccaggc gggccattta ccgtcattga
     9721 cgtcaatagg gggcgtactt ggcatatgat acacttgatg tactgccaag tgggcagttt
     9781 accgtaaata ctccacccat tgacgtcaat ggaaagtccc tattggcgtt actatgggaa
     9841 catacgtcat tattgacgtc aatgggcggg ggtcgttggg cggtcagcca ggcgggccat
     9901 ttaccgtaag ttatgtaacg cggaactcca tatatgggct atgaactaat gaccccgtaa
     9961 ttgattacta ttaataacta gtcaataatc aatgtcaacg cgtatatctg gcccgtacat
    10021 cgcgaagcag cgcaaaacgc ctaaccctaa gcagattctt catgcaattg tcggtcaagc
    10081 cttgccttgt tgtagcttaa attttgctcg cgcactactc agcgacctcc aacacacaag
    10141 cagggagcag atactggctt aactatgcgg catcagagca gattgtactg agagtgcacc
    10201 ataggggatc gggagatctc ccgatccgtc gacgtcaggt ggcacttttc ggggaaatgt
    10261 gcgcggaacc cctatttgtt tatttttcta aatacattca aatatgtatc cgctcatgag
    10321 acaataaccc tgataaatgc ttcaataata ttgaaaaagg aagagtatga gtattcaaca
    10381 tttccgtgtc gcccttattc ccttttttgc ggcattttgc cttcctgttt ttgctcaccc
    10441 agaaacgctg gtgaaagtaa aagatgctga agatcagttg ggtgcacgag tgggttacat
    10501 cgaactggat ctcaacagcg gtaagatcct tgagagtttt cgccccgaag aacgttttcc
    10561 aatgatgagc acttttaaag ttctgctatg tggcgcggta ttatcccgta ttgacgccgg
    10621 gcaagagcaa ctcggtcgcc gcatacacta ttctcagaat gacttggttg agtactcacc
    10681 agtcacagaa aagcatctta cggatggcat gacagtaaga gaattatgca gtgctgccat
    10741 aaccatgagt gataacactg cggccaactt acttctgaca acgatcggag gaccgaagga
    10801 gctaaccgct tttttgcaca acatggggga tcatgtaact cgccttgatc gttgggaacc
    10861 ggagctgaat gaagccatac caaacgacga gcgtgacacc acgatgcctg tagcaatggc
    10921 aacaacgttg cgcaaactat taactggcga actacttact ctagcttccc ggcaacaatt
    10981 aatagactgg atggaggcgg ataaagttgc aggaccactt ctgcgctcgg cccttccggc
    11041 tggctggttt attgctgata aatctggagc cggtgagcgt gggtctcgcg gtatcattgc
    11101 agcactgggg ccagatggta agccctcccg tatcgtagtt atctacacga cggggagtca
    11161 ggcaactatg gatgaacgaa atagacagat cgctgagata ggtgcctcac tgattaagca
    11221 ttggtaactg tcagaccaag tttactcata tatactttag attgatttaa aacttcattt
    11281 ttaatttaaa aggatctagg tgaagatcct ttttgataat ctcatgacca aaatccctta
    11341 acgtgagttt tcgttccact gagcgtcaga ccccgtagaa aagatcaaag gatcttcttg
    11401 agatcctttt tttctgcgcg taatctgctg cttgcaaaca aaaaaaccac cgctaccagc
    11461 ggtggtttgt ttgccggatc aagagctacc aactcttttt ccgaaggtaa ctggcttcag
    11521 cagagcgcag ataccaaata ctgttcttct agtgtagccg tagttaggcc accacttcaa
    11581 gaactctgta gcaccgccta catacctcgc tctgctaatc ctgttaccag tggctgctgc
    11641 cagtggcgat aagtcgtgtc ttaccgggtt ggactcaaga cgatagttac cggataaggc
    11701 gcagcggtcg ggctgaacgg ggggttcgtg cacacagccc agcttggagc gaacgaccta
    11761 caccgaactg agatacctac agcgtgagct atgagaaagc gccacgcttc ccgaagggag
    11821 aaaggcggac aggtatccgg taagcggcag ggtcggaaca ggagagcgca cgagggagct
    11881 tccaggggga aacgcctggt atctttatag tcctgtcggg tttcgccacc tctgacttga
    11941 gcgtcgattt ttgtgatgct cgtcaggggg gcggagccta tggaaaaacg ccagcaacgc
    12001 ggccttttta cggttcctgg ccttttgctg gccttttgct cacatgttct ttcctgcgtt
    12061 atcccctgat tctgtggata accgtattac cgcctttgag tgagctgata ccgctcgccg
    12121 cagccgaacg accgagcgca gcgagtcagt gagcgaggaa gcggaagagc gcccaatacg
    12181 caaaccgcct ctccccgcgc gttggccgat tcattaatgc agctggcacg acaggtttcc
    12241 cgactggaaa gcgggcagtg agcgcaacgc aattaatgtg agttagctca ctcattaggc
    12301 accccaggct ttacacttta tgcttccggc tcgtatgttg tgtggaattg tgagcggata
    12361 acaatttcac acaggaaaca gctatgacca tgattacgcc aagctctagc tagaggtcga
    12421 cggtatacag acatgataag atacattgat gagtttggac aaaccacaac tagaatgcag
    12481 tgaaaaaaat gctttatttg tgaaatttgt gatgctattg ctttatttgt aaccattata
    12541 agctgcaata aacaagttgg ggtgggcgaa gaactccagc atgagatccc cgcgctggag
    12601 gatcatccag ccggcgtccc ggaaaacgat tccgaagccc aacctttcat agaaggcggc
    12661 ggtggaatcg aaatctcgta gcacgtgtca gtcctgctcc tcggccacga agtgcacgca
    12721 gttgccggcc gggtcgcgca gggcgaactc ccgcccccac ggctgctcgc cgatctcggt
    12781 catggccggc ccggaggcgt cccggaagtt cgtggacacg acctccgacc actcggcgta
    12841 cagctcgtcc aggccgcgca cccacaccca ggccagggtg ttgtccggca ccacctggtc
    12901 ctggaccgcg ctgatgaaca gggtcacgtc gtcccggacc acaccggcga agtcgtcctc
    12961 cacgaagtcc cgggagaacc cgagccggtc ggtccagaac tcgaccgctc cggcgacgtc
    13021 gcgcgcggtg agcaccggaa cggcactggt caacttggcc atggtttagt tcctcacctt
    13081 gtcgtattat actatgccga tatactatgc cgatgattaa ttgtcaacac gtgct
//
