LOCUS       sequence-489388-       13013 bp    DNA     circular SYN 15-OCT-2025
DEFINITION  Knockout of human HMBS.
ACCESSION   .
VERSION     .
KEYWORDS    .
SOURCE      synthetic DNA construct
  ORGANISM  synthetic DNA construct
REFERENCE   1  (bases 1 to 13013)
  AUTHORS   Harhai M, Foged MM, Zarges C, Landoni JC, Chollet S, Simonelli M, 
            Recazens E, Lisci M, Laban N, Manley S, Riemer J, Lopez-Escamez JA, 
            Lysakowski A, Jourdain AA
  TITLE     An updated inventory of genes essential for oxidative 
            phosphorylation identifies a mitochondrial origin in familial 
            Meniere's disease.
  JOURNAL   Cell Rep. 2025 Jul 25;44(8):116069. doi: 
            10.1016/j.celrep.2025.116069.
  PUBMED    40714634
REFERENCE   2  (bases 1 to 13013)
  AUTHORS   .
  TITLE     Direct Submission
  JOURNAL   Exported Oct 15, 2025 from SnapGene Server 8.0.1
            https://www.snapgene.com
COMMENT     SGRef: number: 1; type: "Journal Article"; journalName: "Cell Rep.";
            date: "2025-07-25"; volume: "44(8):116069. doi"; pages: " 
            10.1016/j.celrep.2025.116069"
COMMENT     Sequence Label: pLentiCRISPR V2 HMBS_sg2
FEATURES             Location/Qualifiers
     source          1..13013
                     /mol_type="other DNA"
                     /organism="synthetic DNA construct"
     primer_bind     464..484
                     /label=Puro-F
                     /note="Puromycin resistance gene, forward primer"
     misc_feature    583..1171
                     /label=WPRE
                     /note="woodchuck hepatitis virus posttranscriptional 
                     regulatory element"
     primer_bind     complement(636..656)
                     /label=WPRE-R
                     /note="WPRE, reverse primer"
     CDS             complement(1054..1065)
                     /codon_start=1
                     /product="Factor Xa recognition and cleavage site"
                     /label=Factor Xa site
                     /translation="IEGR"
     LTR             1243..1476
                     /label=3' LTR (Delta-U3)
                     /note="self-inactivating 3' long terminal repeat (LTR) from
                     HIV-1"
     primer_bind     complement(1502..1519)
                     /label=BGH-rev
                     /note="Bovine growth hormone terminator, reverse primer. 
                     Also called BGH reverse"
     polyA_signal    1508..1732
                     /label=bGH poly(A) signal
                     /note="bovine growth hormone polyadenylation signal"
     rep_origin      1778..2206
                     /direction=RIGHT
                     /label=f1 ori
                     /note="f1 bacteriophage origin of replication; arrow 
                     indicates direction of (+) strand synthesis"
     primer_bind     complement(1865..1884)
                     /label=F1ori-R
                     /note="F1 origin, reverse primer"
     primer_bind     2075..2096
                     /label=F1ori-F
                     /note="F1 origin, forward primer"
     primer_bind     complement(2215..2235)
                     /label=pBABE 3'
                     /note="SV40 enhancer, reverse primer for pBABE vectors"
     promoter        2220..2549
                     /label=SV40 promoter
                     /note="SV40 enhancer and early promoter"
     rep_origin      2400..2535
                     /label=SV40 ori
                     /note="SV40 origin of replication"
     primer_bind     2462..2481
                     /label=SV40pro-F
                     /note="SV40 promoter/origin, forward primer"
     promoter        2597..2644
                     /label=EM7 promoter
                     /note="synthetic bacterial promoter "
     CDS             2663..3037
                     /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    3167..3288
                     /label=SV40 poly(A) signal
                     /note="SV40 polyadenylation signal"
     primer_bind     complement(3204..3223)
                     /label=SV40pA-R
                     /note="SV40 polyA, reverse primer"
     primer_bind     3258..3277
                     /label=EBV-rev
                     /note="SV40 polyA terminator, reverse primer"
     primer_bind     complement(3337..3353)
                     /label=M13 rev
                     /note="common sequencing primer, one of multiple similar 
                     variants"
     primer_bind     complement(3337..3353)
                     /label=M13 Reverse
                     /note="In lacZ gene. Also called M13-rev"
     primer_bind     complement(3350..3372)
                     /label=M13/pUC Reverse
                     /note="In lacZ gene"
     protein_bind    3361..3377
                     /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(3385..3391,3392..3409,3410..3415))
                     /label=lac promoter
                     /note="promoter for the E. coli lac operon"
     protein_bind    3430..3451
                     /label=CAP binding site
                     /bound_moiety="E. coli catabolite activator protein"
                     /note="CAP binding activates transcription in the presence 
                     of cAMP."
     primer_bind     complement(3568..3585)
                     /label=L4440
                     /note="L4440 vector, forward primer"
     rep_origin      complement(3739..4327)
                     /direction=LEFT
                     /label=ori
                     /note="high-copy-number ColE1/pMB1/pBR322/pUC origin of 
                     replication"
     primer_bind     complement(3819..3838)
                     /label=pBR322ori-F
                     /note="pBR322 origin, forward primer"
     CDS             complement(join(4498..5289,5290..5358))
                     /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     5121..5140
                     /label=Amp-R
                     /note="Ampicillin resistance gene, reverse primer"
     promoter        complement(5359..5463)
                     /gene="bla"
                     /label=AmpR promoter
     primer_bind     complement(5538..5557)
                     /label=pRS-marker
                     /note="pRS vectors, use to sequence yeast selectable 
                     marker"
     enhancer        5729..6108
                     /label=CMV enhancer
                     /note="human cytomegalovirus immediate early enhancer"
     promoter        6110..6308
                     /label=CMV promoter
                     /note="human cytomegalovirus (CMV) immediate early 
                     promoter"
     primer_bind     6263..6283
                     /label=CMV-F
                     /note="Human CMV immediate early promoter, forward primer"
     LTR             6326..6506
                     /label=5' LTR (truncated)
                     /note="truncated 5' long terminal repeat (LTR) from HIV-1"
     misc_feature    6553..6678
                     /label=HIV-1 Psi
                     /note="packaging signal of human immunodeficiency virus 
                     type 1"
     misc_feature    7171..7404
                     /label=RRE
                     /note="The Rev response element (RRE) of HIV-1 allows for 
                     Rev-dependent mRNA export from the nucleus to the 
                     cytoplasm."
     CDS             7589..7633
                     /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    7931..8048
                     /label=cPPT/CTS
                     /note="central polypurine tract and central termination 
                     sequence of HIV-1"
     promoter        8099..8347
                     /label=U6 promoter
                     /note="RNA polymerase III promoter for human U6 snRNA 
                     (Domitrovich & Kunkel, 2003)"
     primer_bind     8099..8119
                     /label=hU6-F
                     /note="Human U6 promoter, forward primer"
     primer_bind     8270..8289
                     /label=LKO.1 5'
                     /note="Human U6 promoter, forward primer"
     misc_RNA        8369..8444
                     /label=gRNA scaffold
                     /note="guide RNA scaffold for the Streptococcus pyogenes 
                     CRISPR/Cas9 system"
     promoter        8506..8717
                     /label=EF-1-alpha core promoter
                     /note="core promoter for human elongation factor 
                     EF-1-alpha"
     regulatory      8736..8745
                     /label=Kozak sequence
                     /note="vertebrate consensus sequence for strong initiation 
                     of translation (Kozak, 1987)"
                     /regulatory_class="other"
     CDS             8742..12845
                     /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             8742..12845
                     /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             12846..12893
                     /codon_start=1
                     /product="bipartite nuclear localization signal from 
                     nucleoplasmin"
                     /label=nucleoplasmin NLS
                     /translation="KRPAATKKAGQAKKKK"
     CDS             12894..12917
                     /codon_start=1
                     /product="FLAG(R) epitope tag, followed by an enterokinase 
                     cleavage site"
                     /label=FLAG
                     /translation="DYKDDDDK"
     CDS             12927..12983
                     /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             join(12984..13013,1..567)
                     /codon_start=1
                     /gene="pac from Streptomyces alboniger"
                     /product="puromycin N-acetyltransferase"
                     /label=PuroR
                     /note="confers resistance to puromycin"
                     /translation="TEYKPTVRLATRDDVPRAVRTLAAAFADYPATRHTVDPDRHIERV
                     TELQELFLTRVGLDIGKVWVADDGAAVAVWTTPESVEAGAVFAEIGPRMAELSGSRLAA
                     QQQMEGLLAPHRPKEPAWFLATVGVSPDHQGKGLGSAVVLPGVEAAERAGVPAFLETSA
                     PRNLPFYERLGFTVTADVEVPEGPRTWCMTRKPGA"
ORIGIN
        1 acccgcgacg acgtccccag ggccgtacgc accctcgccg ccgcgttcgc cgactacccc
       61 gccacgcgcc acaccgtcga tccggaccgc cacatcgagc gggtcaccga gctgcaagaa
      121 ctcttcctca cgcgcgtcgg gctcgacatc ggcaaggtgt gggtcgcgga cgacggcgcc
      181 gcggtggcgg tctggaccac gccggagagc gtcgaagcgg gggcggtgtt cgccgagatc
      241 ggcccgcgca tggccgagtt gagcggttcc cggctggccg cgcagcaaca gatggaaggc
      301 ctcctggcgc cgcaccggcc caaggagccc gcgtggttcc tggccaccgt cggagtctcg
      361 cccgaccacc agggcaaggg tctgggcagc gccgtcgtgc tccccggagt ggaggcggcc
      421 gagcgcgccg gggtgcccgc cttcctggag acctccgcgc cccgcaacct ccccttctac
      481 gagcggctcg gcttcaccgt caccgccgac gtcgaggtgc ccgaaggacc gcgcacctgg
      541 tgcatgaccc gcaagcccgg tgcctgaacg cgttaagtcg acaatcaacc tctggattac
      601 aaaatttgtg aaagattgac tggtattctt aactatgttg ctccttttac gctatgtgga
      661 tacgctgctt taatgccttt gtatcatgct attgcttccc gtatggcttt cattttctcc
      721 tccttgtata aatcctggtt gctgtctctt tatgaggagt tgtggcccgt tgtcaggcaa
      781 cgtggcgtgg tgtgcactgt gtttgctgac gcaaccccca ctggttgggg cattgccacc
      841 acctgtcagc tcctttccgg gactttcgct ttccccctcc ctattgccac ggcggaactc
      901 atcgccgcct gccttgcccg ctgctggaca ggggctcggc tgttgggcac tgacaattcc
      961 gtggtgttgt cggggaaatc atcgtccttt ccttggctgc tcgcctgtgt tgccacctgg
     1021 attctgcgcg ggacgtcctt ctgctacgtc ccttcggccc tcaatccagc ggaccttcct
     1081 tcccgcggcc tgctgccggc tctgcggcct cttccgcgtc ttcgccttcg ccctcagacg
     1141 agtcggatct ccctttgggc cgcctccccg cgtcgacttt aagaccaatg acttacaagg
     1201 cagctgtaga tcttagccac tttttaaaag aaaagggggg actggaaggg ctaattcact
     1261 cccaacgaag acaagatctg ctttttgctt gtactgggtc tctctggtta gaccagatct
     1321 gagcctggga gctctctggc taactaggga acccactgct taagcctcaa taaagcttgc
     1381 cttgagtgct tcaagtagtg tgtgcccgtc tgttgtgtga ctctggtaac tagagatccc
     1441 tcagaccctt ttagtcagtg tggaaaatct ctagcagggc ccgtttaaac ccgctgatca
     1501 gcctcgactg tgccttctag ttgccagcca tctgttgttt gcccctcccc cgtgccttcc
     1561 ttgaccctgg aaggtgccac tcccactgtc ctttcctaat aaaatgagga aattgcatcg
     1621 cattgtctga gtaggtgtca ttctattctg gggggtgggg tggggcagga cagcaagggg
     1681 gaggattggg aagacaatag caggcatgct ggggatgcgg tgggctctat ggcttctgag
     1741 gcggaaagaa ccagctgggg ctctaggggg tatccccacg cgccctgtag cggcgcatta
     1801 agcgcggcgg gtgtggtggt tacgcgcagc gtgaccgcta cacttgccag cgccctagcg
     1861 cccgctcctt tcgctttctt cccttccttt ctcgccacgt tcgccggctt tccccgtcaa
     1921 gctctaaatc gggggctccc tttagggttc cgatttagtg ctttacggca cctcgacccc
     1981 aaaaaacttg attagggtga tggttcacgt agtgggccat cgccctgata gacggttttt
     2041 cgccctttga cgttggagtc cacgttcttt aatagtggac tcttgttcca aactggaaca
     2101 acactcaacc ctatctcggt ctattctttt gatttataag ggattttgcc gatttcggcc
     2161 tattggttaa aaaatgagct gatttaacaa aaatttaacg cgaattaatt ctgtggaatg
     2221 tgtgtcagtt agggtgtgga aagtccccag gctccccagc aggcagaagt atgcaaagca
     2281 tgcatctcaa ttagtcagca accaggtgtg gaaagtcccc aggctcccca gcaggcagaa
     2341 gtatgcaaag catgcatctc aattagtcag caaccatagt cccgccccta actccgccca
     2401 tcccgcccct aactccgccc agttccgccc attctccgcc ccatggctga ctaatttttt
     2461 ttatttatgc agaggccgag gccgcctctg cctctgagct attccagaag tagtgaggag
     2521 gcttttttgg aggcctaggc ttttgcaaaa agctcccggg agcttgtata tccattttcg
     2581 gatctgatca gcacgtgttg acaattaatc atcggcatag tatatcggca tagtataata
     2641 cgacaaggtg aggaactaaa ccatggccaa gttgaccagt gccgttccgg tgctcaccgc
     2701 gcgcgacgtc gccggagcgg tcgagttctg gaccgaccgg ctcgggttct cccgggactt
     2761 cgtggaggac gacttcgccg gtgtggtccg ggacgacgtg accctgttca tcagcgcggt
     2821 ccaggaccag gtggtgccgg acaacaccct ggcctgggtg tgggtgcgcg gcctggacga
     2881 gctgtacgcc gagtggtcgg aggtcgtgtc cacgaacttc cgggacgcct ccgggccggc
     2941 catgaccgag atcggcgagc agccgtgggg gcgggagttc gccctgcgcg acccggccgg
     3001 caactgcgtg cacttcgtgg ccgaggagca ggactgacac gtgctacgag atttcgattc
     3061 caccgccgcc ttctatgaaa ggttgggctt cggaatcgtt ttccgggacg ccggctggat
     3121 gatcctccag cgcggggatc tcatgctgga gttcttcgcc caccccaact tgtttattgc
     3181 agcttataat ggttacaaat aaagcaatag catcacaaat ttcacaaata aagcattttt
     3241 ttcactgcat tctagttgtg gtttgtccaa actcatcaat gtatcttatc atgtctgtat
     3301 accgtcgacc tctagctaga gcttggcgta atcatggtca tagctgtttc ctgtgtgaaa
     3361 ttgttatccg ctcacaattc cacacaacat acgagccgga agcataaagt gtaaagcctg
     3421 gggtgcctaa tgagtgagct aactcacatt aattgcgttg cgctcactgc ccgctttcca
     3481 gtcgggaaac ctgtcgtgcc agctgcatta atgaatcggc caacgcgcgg ggagaggcgg
     3541 tttgcgtatt gggcgctctt ccgcttcctc gctcactgac tcgctgcgct cggtcgttcg
     3601 gctgcggcga gcggtatcag ctcactcaaa ggcggtaata cggttatcca cagaatcagg
     3661 ggataacgca ggaaagaaca tgtgagcaaa aggccagcaa aaggccagga accgtaaaaa
     3721 ggccgcgttg ctggcgtttt tccataggct ccgcccccct gacgagcatc acaaaaatcg
     3781 acgctcaagt cagaggtggc gaaacccgac aggactataa agataccagg cgtttccccc
     3841 tggaagctcc ctcgtgcgct ctcctgttcc gaccctgccg cttaccggat acctgtccgc
     3901 ctttctccct tcgggaagcg tggcgctttc tcatagctca cgctgtaggt atctcagttc
     3961 ggtgtaggtc gttcgctcca agctgggctg tgtgcacgaa ccccccgttc agcccgaccg
     4021 ctgcgcctta tccggtaact atcgtcttga gtccaacccg gtaagacacg acttatcgcc
     4081 actggcagca gccactggta acaggattag cagagcgagg tatgtaggcg gtgctacaga
     4141 gttcttgaag tggtggccta actacggcta cactagaaga acagtatttg gtatctgcgc
     4201 tctgctgaag ccagttacct tcggaaaaag agttggtagc tcttgatccg gcaaacaaac
     4261 caccgctggt agcggtggtt tttttgtttg caagcagcag attacgcgca gaaaaaaagg
     4321 atctcaagaa gatcctttga tcttttctac ggggtctgac gctcagtgga acgaaaactc
     4381 acgttaaggg attttggtca tgagattatc aaaaaggatc ttcacctaga tccttttaaa
     4441 ttaaaaatga agttttaaat caatctaaag tatatatgag taaacttggt ctgacagtta
     4501 ccaatgctta atcagtgagg cacctatctc agcgatctgt ctatttcgtt catccatagt
     4561 tgcctgactc cccgtcgtgt agataactac gatacgggag ggcttaccat ctggccccag
     4621 tgctgcaatg ataccgcgag acccacgctc accggctcca gatttatcag caataaacca
     4681 gccagccgga agggccgagc gcagaagtgg tcctgcaact ttatccgcct ccatccagtc
     4741 tattaattgt tgccgggaag ctagagtaag tagttcgcca gttaatagtt tgcgcaacgt
     4801 tgttgccatt gctacaggca tcgtggtgtc acgctcgtcg tttggtatgg cttcattcag
     4861 ctccggttcc caacgatcaa ggcgagttac atgatccccc atgttgtgca aaaaagcggt
     4921 tagctccttc ggtcctccga tcgttgtcag aagtaagttg gccgcagtgt tatcactcat
     4981 ggttatggca gcactgcata attctcttac tgtcatgcca tccgtaagat gcttttctgt
     5041 gactggtgag tactcaacca agtcattctg agaatagtgt atgcggcgac cgagttgctc
     5101 ttgcccggcg tcaatacggg ataataccgc gccacatagc agaactttaa aagtgctcat
     5161 cattggaaaa cgttcttcgg ggcgaaaact ctcaaggatc ttaccgctgt tgagatccag
     5221 ttcgatgtaa cccactcgtg cacccaactg atcttcagca tcttttactt tcaccagcgt
     5281 ttctgggtga gcaaaaacag gaaggcaaaa tgccgcaaaa aagggaataa gggcgacacg
     5341 gaaatgttga atactcatac tcttcctttt tcaatattat tgaagcattt atcagggtta
     5401 ttgtctcatg agcggataca tatttgaatg tatttagaaa aataaacaaa taggggttcc
     5461 gcgcacattt ccccgaaaag tgccacctga cgtcgacgga tcgggagatc tcccgatccc
     5521 ctatggtgca ctctcagtac aatctgctct gatgccgcat agttaagcca gtatctgctc
     5581 cctgcttgtg tgttggaggt cgctgagtag tgcgcgagca aaatttaagc tacaacaagg
     5641 caaggcttga ccgacaattg catgaagaat ctgcttaggg ttaggcgttt tgcgctgctt
     5701 cgcgatgtac gggccagata tacgcgttga cattgattat tgactagtta ttaatagtaa
     5761 tcaattacgg ggtcattagt tcatagccca tatatggagt tccgcgttac ataacttacg
     5821 gtaaatggcc cgcctggctg accgcccaac gacccccgcc cattgacgtc aataatgacg
     5881 tatgttccca tagtaacgcc aatagggact ttccattgac gtcaatgggt ggagtattta
     5941 cggtaaactg cccacttggc agtacatcaa gtgtatcata tgccaagtac gccccctatt
     6001 gacgtcaatg acggtaaatg gcccgcctgg cattatgccc agtacatgac cttatgggac
     6061 tttcctactt ggcagtacat ctacgtatta gtcatcgcta ttaccatggt gatgcggttt
     6121 tggcagtaca tcaatgggcg tggatagcgg tttgactcac ggggatttcc aagtctccac
     6181 cccattgacg tcaatgggag tttgttttgg caccaaaatc aacgggactt tccaaaatgt
     6241 cgtaacaact ccgccccatt gacgcaaatg ggcggtaggc gtgtacggtg ggaggtctat
     6301 ataagcagcg cgttttgcct gtactgggtc tctctggtta gaccagatct gagcctggga
     6361 gctctctggc taactaggga acccactgct taagcctcaa taaagcttgc cttgagtgct
     6421 tcaagtagtg tgtgcccgtc tgttgtgtga ctctggtaac tagagatccc tcagaccctt
     6481 ttagtcagtg tggaaaatct ctagcagtgg cgcccgaaca gggacttgaa agcgaaaggg
     6541 aaaccagagg agctctctcg acgcaggact cggcttgctg aagcgcgcac ggcaagaggc
     6601 gaggggcggc gactggtgag tacgccaaaa attttgacta gcggaggcta gaaggagaga
     6661 gatgggtgcg agagcgtcag tattaagcgg gggagaatta gatcgcgatg ggaaaaaatt
     6721 cggttaaggc cagggggaaa gaaaaaatat aaattaaaac atatagtatg ggcaagcagg
     6781 gagctagaac gattcgcagt taatcctggc ctgttagaaa catcagaagg ctgtagacaa
     6841 atactgggac agctacaacc atcccttcag acaggatcag aagaacttag atcattatat
     6901 aatacagtag caaccctcta ttgtgtgcat caaaggatag agataaaaga caccaaggaa
     6961 gctttagaca agatagagga agagcaaaac aaaagtaaga ccaccgcaca gcaagcggcc
     7021 gctgatcttc agacctggag gaggagatat gagggacaat tggagaagtg aattatataa
     7081 atataaagta gtaaaaattg aaccattagg agtagcaccc accaaggcaa agagaagagt
     7141 ggtgcagaga gaaaaaagag cagtgggaat aggagctttg ttccttgggt tcttgggagc
     7201 agcaggaagc actatgggcg cagcgtcaat gacgctgacg gtacaggcca gacaattatt
     7261 gtctggtata gtgcagcagc agaacaattt gctgagggct attgaggcgc aacagcatct
     7321 gttgcaactc acagtctggg gcatcaagca gctccaggca agaatcctgg ctgtggaaag
     7381 atacctaaag gatcaacagc tcctggggat ttggggttgc tctggaaaac tcatttgcac
     7441 cactgctgtg ccttggaatg ctagttggag taataaatct ctggaacaga tttggaatca
     7501 cacgacctgg atggagtggg acagagaaat taacaattac acaagcttaa tacactcctt
     7561 aattgaagaa tcgcaaaacc agcaagaaaa gaatgaacaa gaattattgg aattagataa
     7621 atgggcaagt ttgtggaatt ggtttaacat aacaaattgg ctgtggtata taaaattatt
     7681 cataatgata gtaggaggct tggtaggttt aagaatagtt tttgctgtac tttctatagt
     7741 gaatagagtt aggcagggat attcaccatt atcgtttcag acccacctcc caaccccgag
     7801 gggacccgac aggcccgaag gaatagaaga agaaggtgga gagagagaca gagacagatc
     7861 cattcgatta gtgaacggat cggcactgcg tgcgccaatt ctgcagacaa atggcagtat
     7921 tcatccacaa ttttaaaaga aaagggggga ttggggggta cagtgcaggg gaaagaatag
     7981 tagacataat agcaacagac atacaaacta aagaattaca aaaacaaatt acaaaaattc
     8041 aaaattttcg ggtttattac agggacagca gagatccagt ttggttaatt aaggtaccga
     8101 gggcctattt cccatgattc cttcatattt gcatatacga tacaaggctg ttagagagat
     8161 aattagaatt aatttgactg taaacacaaa gatattagta caaaatacgt gacgtagaaa
     8221 gtaataattt cttgggtagt ttgcagtttt aaaattatgt tttaaaatgg actatcatat
     8281 gcttaccgta acttgaaagt atttcgattt cttggcttta tatatcttgt ggaaaggacg
     8341 aaacaccgat caagaatctt gtcccctggt tttagagcta gaaatagcaa gttaaaataa
     8401 ggctagtccg ttatcaactt gaaaaagtgg caccgagtcg gtgctttttt gaattcgcta
     8461 gctaggtctt gaaaggagtg ggaattggct ccggtgcccg tcagtgggca gagcgcacat
     8521 cgcccacagt ccccgagaag ttggggggag gggtcggcaa ttgatccggt gcctagagaa
     8581 ggtggcgcgg ggtaaactgg gaaagtgatg tcgtgtactg gctccgcctt tttcccgagg
     8641 gtgggggaga accgtatata agtgcagtag tcgccgtgaa cgttcttttt cgcaacgggt
     8701 ttgccgccag aacacaggac cggttctaga gcgctgccac catggacaag aagtacagca
     8761 tcggcctgga catcggcacc aactctgtgg gctgggccgt gatcaccgac gagtacaagg
     8821 tgcccagcaa gaaattcaag gtgctgggca acaccgaccg gcacagcatc aagaagaacc
     8881 tgatcggagc cctgctgttc gacagcggcg aaacagccga ggccacccgg ctgaagagaa
     8941 ccgccagaag aagatacacc agacggaaga accggatctg ctatctgcaa gagatcttca
     9001 gcaacgagat ggccaaggtg gacgacagct tcttccacag actggaagag tccttcctgg
     9061 tggaagagga taagaagcac gagcggcacc ccatcttcgg caacatcgtg gacgaggtgg
     9121 cctaccacga gaagtacccc accatctacc acctgagaaa gaaactggtg gacagcaccg
     9181 acaaggccga cctgcggctg atctatctgg ccctggccca catgatcaag ttccggggcc
     9241 acttcctgat cgagggcgac ctgaaccccg acaacagcga cgtggacaag ctgttcatcc
     9301 agctggtgca gacctacaac cagctgttcg aggaaaaccc catcaacgcc agcggcgtgg
     9361 acgccaaggc catcctgtct gccagactga gcaagagcag acggctggaa aatctgatcg
     9421 cccagctgcc cggcgagaag aagaatggcc tgttcggaaa cctgattgcc ctgagcctgg
     9481 gcctgacccc caacttcaag agcaacttcg acctggccga ggatgccaaa ctgcagctga
     9541 gcaaggacac ctacgacgac gacctggaca acctgctggc ccagatcggc gaccagtacg
     9601 ccgacctgtt tctggccgcc aagaacctgt ccgacgccat cctgctgagc gacatcctga
     9661 gagtgaacac cgagatcacc aaggcccccc tgagcgcctc tatgatcaag agatacgacg
     9721 agcaccacca ggacctgacc ctgctgaaag ctctcgtgcg gcagcagctg cctgagaagt
     9781 acaaagagat tttcttcgac cagagcaaga acggctacgc cggctacatt gacggcggag
     9841 ccagccagga agagttctac aagttcatca agcccatcct ggaaaagatg gacggcaccg
     9901 aggaactgct cgtgaagctg aacagagagg acctgctgcg gaagcagcgg accttcgaca
     9961 acggcagcat cccccaccag atccacctgg gagagctgca cgccattctg cggcggcagg
    10021 aagattttta cccattcctg aaggacaacc gggaaaagat cgagaagatc ctgaccttcc
    10081 gcatccccta ctacgtgggc cctctggcca ggggaaacag cagattcgcc tggatgacca
    10141 gaaagagcga ggaaaccatc accccctgga acttcgagga agtggtggac aagggcgctt
    10201 ccgcccagag cttcatcgag cggatgacca acttcgataa gaacctgccc aacgagaagg
    10261 tgctgcccaa gcacagcctg ctgtacgagt acttcaccgt gtataacgag ctgaccaaag
    10321 tgaaatacgt gaccgaggga atgagaaagc ccgccttcct gagcggcgag cagaaaaagg
    10381 ccatcgtgga cctgctgttc aagaccaacc ggaaagtgac cgtgaagcag ctgaaagagg
    10441 actacttcaa gaaaatcgag tgcttcgact ccgtggaaat ctccggcgtg gaagatcggt
    10501 tcaacgcctc cctgggcaca taccacgatc tgctgaaaat tatcaaggac aaggacttcc
    10561 tggacaatga ggaaaacgag gacattctgg aagatatcgt gctgaccctg acactgtttg
    10621 aggacagaga gatgatcgag gaacggctga aaacctatgc ccacctgttc gacgacaaag
    10681 tgatgaagca gctgaagcgg cggagataca ccggctgggg caggctgagc cggaagctga
    10741 tcaacggcat ccgggacaag cagtccggca agacaatcct ggatttcctg aagtccgacg
    10801 gcttcgccaa cagaaacttc atgcagctga tccacgacga cagcctgacc tttaaagagg
    10861 acatccagaa agcccaggtg tccggccagg gcgatagcct gcacgagcac attgccaatc
    10921 tggccggcag ccccgccatt aagaagggca tcctgcagac agtgaaggtg gtggacgagc
    10981 tcgtgaaagt gatgggccgg cacaagcccg agaacatcgt gatcgaaatg gccagagaga
    11041 accagaccac ccagaaggga cagaagaaca gccgcgagag aatgaagcgg atcgaagagg
    11101 gcatcaaaga gctgggcagc cagatcctga aagaacaccc cgtggaaaac acccagctgc
    11161 agaacgagaa gctgtacctg tactacctgc agaatgggcg ggatatgtac gtggaccagg
    11221 aactggacat caaccggctg tccgactacg atgtggacca tatcgtgcct cagagctttc
    11281 tgaaggacga ctccatcgac aacaaggtgc tgaccagaag cgacaagaac cggggcaaga
    11341 gcgacaacgt gccctccgaa gaggtcgtga agaagatgaa gaactactgg cggcagctgc
    11401 tgaacgccaa gctgattacc cagagaaagt tcgacaatct gaccaaggcc gagagaggcg
    11461 gcctgagcga actggataag gccggcttca tcaagagaca gctggtggaa acccggcaga
    11521 tcacaaagca cgtggcacag atcctggact cccggatgaa cactaagtac gacgagaatg
    11581 acaagctgat ccgggaagtg aaagtgatca ccctgaagtc caagctggtg tccgatttcc
    11641 ggaaggattt ccagttttac aaagtgcgcg agatcaacaa ctaccaccac gcccacgacg
    11701 cctacctgaa cgccgtcgtg ggaaccgccc tgatcaaaaa gtaccctaag ctggaaagcg
    11761 agttcgtgta cggcgactac aaggtgtacg acgtgcggaa gatgatcgcc aagagcgagc
    11821 aggaaatcgg caaggctacc gccaagtact tcttctacag caacatcatg aactttttca
    11881 agaccgagat taccctggcc aacggcgaga tccggaagcg gcctctgatc gagacaaacg
    11941 gcgaaaccgg ggagatcgtg tgggataagg gccgggattt tgccaccgtg cggaaagtgc
    12001 tgagcatgcc ccaagtgaat atcgtgaaaa agaccgaggt gcagacaggc ggcttcagca
    12061 aagagtctat cctgcccaag aggaacagcg ataagctgat cgccagaaag aaggactggg
    12121 accctaagaa gtacggcggc ttcgacagcc ccaccgtggc ctattctgtg ctggtggtgg
    12181 ccaaagtgga aaagggcaag tccaagaaac tgaagagtgt gaaagagctg ctggggatca
    12241 ccatcatgga aagaagcagc ttcgagaaga atcccatcga ctttctggaa gccaagggct
    12301 acaaagaagt gaaaaaggac ctgatcatca agctgcctaa gtactccctg ttcgagctgg
    12361 aaaacggccg gaagagaatg ctggcctctg ccggcgaact gcagaaggga aacgaactgg
    12421 ccctgccctc caaatatgtg aacttcctgt acctggccag ccactatgag aagctgaagg
    12481 gctcccccga ggataatgag cagaaacagc tgtttgtgga acagcacaag cactacctgg
    12541 acgagatcat cgagcagatc agcgagttct ccaagagagt gatcctggcc gacgctaatc
    12601 tggacaaagt gctgtccgcc tacaacaagc accgggataa gcccatcaga gagcaggccg
    12661 agaatatcat ccacctgttt accctgacca atctgggagc ccctgccgcc ttcaagtact
    12721 ttgacaccac catcgaccgg aagaggtaca ccagcaccaa agaggtgctg gacgccaccc
    12781 tgatccacca gagcatcacc ggcctgtacg agacacggat cgacctgtct cagctgggag
    12841 gcgacaagcg acctgccgcc acaaagaagg ctggacaggc taagaagaag aaagattaca
    12901 aagacgatga cgataaggga tccggcgcaa caaacttctc tctgctgaaa caagccggag
    12961 atgtcgaaga gaatcctgga ccgaccgagt acaagcccac ggtgcgcctc gcc
//
