LOCUS       sequence-489386-       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_sg1
FEATURES             Location/Qualifiers
     source          1..13013
                     /mol_type="other DNA"
                     /organism="synthetic DNA construct"
     CDS             complement(164..208)
                     /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    393..626
                     /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    1119..1244
                     /label=HIV-1 Psi
                     /note="packaging signal of human immunodeficiency virus 
                     type 1"
     LTR             1291..1471
                     /label=5' LTR (truncated)
                     /note="truncated 5' long terminal repeat (LTR) from HIV-1"
     promoter        complement(1489..1687)
                     /label=CMV promoter
                     /note="human cytomegalovirus (CMV) immediate early 
                     promoter"
     primer_bind     complement(1514..1534)
                     /label=CMV-F
                     /note="Human CMV immediate early promoter, forward primer"
     enhancer        1689..2068
                     /label=CMV enhancer
                     /note="human cytomegalovirus immediate early enhancer"
     primer_bind     2240..2259
                     /label=pRS-marker
                     /note="pRS vectors, use to sequence yeast selectable 
                     marker"
     promoter        2334..2438
                     /gene="bla"
                     /label=AmpR promoter
     CDS             join(2439..2507,2508..3299)
                     /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(2657..2676)
                     /label=Amp-R
                     /note="Ampicillin resistance gene, reverse primer"
     rep_origin      3470..4058
                     /direction=RIGHT
                     /label=ori
                     /note="high-copy-number ColE1/pMB1/pBR322/pUC origin of 
                     replication"
     primer_bind     3959..3978
                     /label=pBR322ori-F
                     /note="pBR322 origin, forward primer"
     primer_bind     4212..4229
                     /label=L4440
                     /note="L4440 vector, forward primer"
     protein_bind    4346..4367
                     /label=CAP binding site
                     /bound_moiety="E. coli catabolite activator protein"
                     /note="CAP binding activates transcription in the presence 
                     of cAMP."
     promoter        join(4382..4387,4388..4405,4406..4412)
                     /label=lac promoter
                     /note="promoter for the E. coli lac operon"
     protein_bind    4420..4436
                     /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     4425..4447
                     /label=M13/pUC Reverse
                     /note="In lacZ gene"
     primer_bind     4444..4460
                     /label=M13 rev
                     /note="common sequencing primer, one of multiple similar 
                     variants"
     primer_bind     4444..4460
                     /label=M13 Reverse
                     /note="In lacZ gene. Also called M13-rev"
     polyA_signal    4509..4630
                     /label=SV40 poly(A) signal
                     /note="SV40 polyadenylation signal"
     primer_bind     complement(4520..4539)
                     /label=EBV-rev
                     /note="SV40 polyA terminator, reverse primer"
     primer_bind     4574..4593
                     /label=SV40pA-R
                     /note="SV40 polyA, reverse primer"
     CDS             complement(4760..5134)
                     /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(5153..5200)
                     /label=EM7 promoter
                     /note="synthetic bacterial promoter "
     promoter        complement(5248..5577)
                     /label=SV40 promoter
                     /note="SV40 enhancer and early promoter"
     rep_origin      5262..5397
                     /label=SV40 ori
                     /note="SV40 origin of replication"
     primer_bind     complement(5316..5335)
                     /label=SV40pro-F
                     /note="SV40 promoter/origin, forward primer"
     primer_bind     5562..5582
                     /label=pBABE 3'
                     /note="SV40 enhancer, reverse primer for pBABE vectors"
     rep_origin      complement(5591..6019)
                     /direction=LEFT
                     /label=f1 ori
                     /note="f1 bacteriophage origin of replication; arrow 
                     indicates direction of (+) strand synthesis"
     primer_bind     complement(5701..5722)
                     /label=F1ori-F
                     /note="F1 origin, forward primer"
     primer_bind     5913..5932
                     /label=F1ori-R
                     /note="F1 origin, reverse primer"
     polyA_signal    6065..6289
                     /label=bGH poly(A) signal
                     /note="bovine growth hormone polyadenylation signal"
     primer_bind     6278..6295
                     /label=BGH-rev
                     /note="Bovine growth hormone terminator, reverse primer. 
                     Also called BGH reverse"
     LTR             6321..6554
                     /label=3' LTR (Delta-U3)
                     /note="self-inactivating 3' long terminal repeat (LTR) from
                     HIV-1"
     misc_feature    6626..7214
                     /label=WPRE
                     /note="woodchuck hepatitis virus posttranscriptional 
                     regulatory element"
     CDS             6732..6743
                     /codon_start=1
                     /product="Factor Xa recognition and cleavage site"
                     /label=Factor Xa site
                     /translation="IEGR"
     primer_bind     7141..7161
                     /label=WPRE-R
                     /note="WPRE, reverse primer"
     CDS             complement(7230..7826)
                     /codon_start=1
                     /gene="pac from Streptomyces alboniger"
                     /product="puromycin N-acetyltransferase"
                     /label=PuroR
                     /note="confers resistance to puromycin"
                     /translation="TEYKPTVRLATRDDVPRAVRTLAAAFADYPATRHTVDPDRHIERV
                     TELQELFLTRVGLDIGKVWVADDGAAVAVWTTPESVEAGAVFAEIGPRMAELSGSRLAA
                     QQQMEGLLAPHRPKEPAWFLATVGVSPDHQGKGLGSAVVLPGVEAAERAGVPAFLETSA
                     PRNLPFYERLGFTVTADVEVPEGPRTWCMTRKPGA"
     primer_bind     complement(7313..7333)
                     /label=Puro-F
                     /note="Puromycin resistance gene, forward primer"
     CDS             complement(7827..7883)
                     /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(7893..7916)
                     /codon_start=1
                     /product="FLAG(R) epitope tag, followed by an enterokinase 
                     cleavage site"
                     /label=FLAG
                     /translation="DYKDDDDK"
     CDS             complement(7917..7964)
                     /codon_start=1
                     /product="bipartite nuclear localization signal from 
                     nucleoplasmin"
                     /label=nucleoplasmin NLS
                     /translation="KRPAATKKAGQAKKKK"
     CDS             complement(7965..12068)
                     /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(7965..12068)
                     /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      12065..12074
                     /label=Kozak sequence
                     /note="vertebrate consensus sequence for strong initiation 
                     of translation (Kozak, 1987)"
                     /regulatory_class="other"
     promoter        complement(12093..12304)
                     /label=EF-1-alpha core promoter
                     /note="core promoter for human elongation factor 
                     EF-1-alpha"
     misc_RNA        complement(12366..12441)
                     /label=gRNA scaffold
                     /note="guide RNA scaffold for the Streptococcus pyogenes 
                     CRISPR/Cas9 system"
     promoter        complement(12463..12711)
                     /label=U6 promoter
                     /note="RNA polymerase III promoter for human U6 snRNA 
                     (Domitrovich & Kunkel, 2003)"
     primer_bind     complement(12521..12540)
                     /label=LKO.1 5'
                     /note="Human U6 promoter, forward primer"
     primer_bind     complement(12691..12711)
                     /label=hU6-F
                     /note="Human U6 promoter, forward primer"
     misc_feature    12762..12879
                     /label=cPPT/CTS
                     /note="central polypurine tract and central termination 
                     sequence of HIV-1"
ORIGIN
        1 gggttgggag gtgggtctga aacgataatg gtgaatatcc ctgcctaact ctattcacta
       61 tagaaagtac agcaaaaact attcttaaac ctaccaagcc tcctactatc attatgaata
      121 attttatata ccacagccaa tttgttatgt taaaccaatt ccacaaactt gcccatttat
      181 ctaattccaa taattcttgt tcattctttt cttgctggtt ttgcgattct tcaattaagg
      241 agtgtattaa gcttgtgtaa ttgttaattt ctctgtccca ctccatccag gtcgtgtgat
      301 tccaaatctg ttccagagat ttattactcc aactagcatt ccaaggcaca gcagtggtgc
      361 aaatgagttt tccagagcaa ccccaaatcc ccaggagctg ttgatccttt aggtatcttt
      421 ccacagccag gattcttgcc tggagctgct tgatgcccca gactgtgagt tgcaacagat
      481 gctgttgcgc ctcaatagcc ctcagcaaat tgttctgctg ctgcactata ccagacaata
      541 attgtctggc ctgtaccgtc agcgtcattg acgctgcgcc catagtgctt cctgctgctc
      601 ccaagaaccc aaggaacaaa gctcctattc ccactgctct tttttctctc tgcaccactc
      661 ttctctttgc cttggtgggt gctactccta atggttcaat ttttactact ttatatttat
      721 ataattcact tctccaattg tccctcatat ctcctcctcc aggtctgaag atcagcggcc
      781 gcttgctgtg cggtggtctt acttttgttt tgctcttcct ctatcttgtc taaagcttcc
      841 ttggtgtctt ttatctctat cctttgatgc acacaataga gggttgctac tgtattatat
      901 aatgatctaa gttcttctga tcctgtctga agggatggtt gtagctgtcc cagtatttgt
      961 ctacagcctt ctgatgtttc taacaggcca ggattaactg cgaatcgttc tagctccctg
     1021 cttgcccata ctatatgttt taatttatat tttttctttc cccctggcct taaccgaatt
     1081 ttttcccatc gcgatctaat tctcccccgc ttaatactga cgctctcgca cccatctctc
     1141 tccttctagc ctccgctagt caaaattttt ggcgtactca ccagtcgccg cccctcgcct
     1201 cttgccgtgc gcgcttcagc aagccgagtc ctgcgtcgag agagctcctc tggtttccct
     1261 ttcgctttca agtccctgtt cgggcgccac tgctagagat tttccacact gactaaaagg
     1321 gtctgaggga tctctagtta ccagagtcac acaacagacg ggcacacact acttgaagca
     1381 ctcaaggcaa gctttattga ggcttaagca gtgggttccc tagttagcca gagagctccc
     1441 aggctcagat ctggtctaac cagagagacc cagtacaggc aaaacgcgct gcttatatag
     1501 acctcccacc gtacacgcct accgcccatt tgcgtcaatg gggcggagtt gttacgacat
     1561 tttggaaagt cccgttgatt ttggtgccaa aacaaactcc cattgacgtc aatggggtgg
     1621 agacttggaa atccccgtga gtcaaaccgc tatccacgcc cattgatgta ctgccaaaac
     1681 cgcatcacca tggtaatagc gatgactaat acgtagatgt actgccaagt aggaaagtcc
     1741 cataaggtca tgtactgggc ataatgccag gcgggccatt taccgtcatt gacgtcaata
     1801 gggggcgtac ttggcatatg atacacttga tgtactgcca agtgggcagt ttaccgtaaa
     1861 tactccaccc attgacgtca atggaaagtc cctattggcg ttactatggg aacatacgtc
     1921 attattgacg tcaatgggcg ggggtcgttg ggcggtcagc caggcgggcc atttaccgta
     1981 agttatgtaa cgcggaactc catatatggg ctatgaacta atgaccccgt aattgattac
     2041 tattaataac tagtcaataa tcaatgtcaa cgcgtatatc tggcccgtac atcgcgaagc
     2101 agcgcaaaac gcctaaccct aagcagattc ttcatgcaat tgtcggtcaa gccttgcctt
     2161 gttgtagctt aaattttgct cgcgcactac tcagcgacct ccaacacaca agcagggagc
     2221 agatactggc ttaactatgc ggcatcagag cagattgtac tgagagtgca ccatagggga
     2281 tcgggagatc tcccgatccg tcgacgtcag gtggcacttt tcggggaaat gtgcgcggaa
     2341 cccctatttg tttatttttc taaatacatt caaatatgta tccgctcatg agacaataac
     2401 cctgataaat gcttcaataa tattgaaaaa ggaagagtat gagtattcaa catttccgtg
     2461 tcgcccttat tccctttttt gcggcatttt gccttcctgt ttttgctcac ccagaaacgc
     2521 tggtgaaagt aaaagatgct gaagatcagt tgggtgcacg agtgggttac atcgaactgg
     2581 atctcaacag cggtaagatc cttgagagtt ttcgccccga agaacgtttt ccaatgatga
     2641 gcacttttaa agttctgcta tgtggcgcgg tattatcccg tattgacgcc gggcaagagc
     2701 aactcggtcg ccgcatacac tattctcaga atgacttggt tgagtactca ccagtcacag
     2761 aaaagcatct tacggatggc atgacagtaa gagaattatg cagtgctgcc ataaccatga
     2821 gtgataacac tgcggccaac ttacttctga caacgatcgg aggaccgaag gagctaaccg
     2881 cttttttgca caacatgggg gatcatgtaa ctcgccttga tcgttgggaa ccggagctga
     2941 atgaagccat accaaacgac gagcgtgaca ccacgatgcc tgtagcaatg gcaacaacgt
     3001 tgcgcaaact attaactggc gaactactta ctctagcttc ccggcaacaa ttaatagact
     3061 ggatggaggc ggataaagtt gcaggaccac ttctgcgctc ggcccttccg gctggctggt
     3121 ttattgctga taaatctgga gccggtgagc gtgggtctcg cggtatcatt gcagcactgg
     3181 ggccagatgg taagccctcc cgtatcgtag ttatctacac gacggggagt caggcaacta
     3241 tggatgaacg aaatagacag atcgctgaga taggtgcctc actgattaag cattggtaac
     3301 tgtcagacca agtttactca tatatacttt agattgattt aaaacttcat ttttaattta
     3361 aaaggatcta ggtgaagatc ctttttgata atctcatgac caaaatccct taacgtgagt
     3421 tttcgttcca ctgagcgtca gaccccgtag aaaagatcaa aggatcttct tgagatcctt
     3481 tttttctgcg cgtaatctgc tgcttgcaaa caaaaaaacc accgctacca gcggtggttt
     3541 gtttgccgga tcaagagcta ccaactcttt ttccgaaggt aactggcttc agcagagcgc
     3601 agataccaaa tactgttctt ctagtgtagc cgtagttagg ccaccacttc aagaactctg
     3661 tagcaccgcc tacatacctc gctctgctaa tcctgttacc agtggctgct gccagtggcg
     3721 ataagtcgtg tcttaccggg ttggactcaa gacgatagtt accggataag gcgcagcggt
     3781 cgggctgaac ggggggttcg tgcacacagc ccagcttgga gcgaacgacc tacaccgaac
     3841 tgagatacct acagcgtgag ctatgagaaa gcgccacgct tcccgaaggg agaaaggcgg
     3901 acaggtatcc ggtaagcggc agggtcggaa caggagagcg cacgagggag cttccagggg
     3961 gaaacgcctg gtatctttat agtcctgtcg ggtttcgcca cctctgactt gagcgtcgat
     4021 ttttgtgatg ctcgtcaggg gggcggagcc tatggaaaaa cgccagcaac gcggcctttt
     4081 tacggttcct ggccttttgc tggccttttg ctcacatgtt ctttcctgcg ttatcccctg
     4141 attctgtgga taaccgtatt accgcctttg agtgagctga taccgctcgc cgcagccgaa
     4201 cgaccgagcg cagcgagtca gtgagcgagg aagcggaaga gcgcccaata cgcaaaccgc
     4261 ctctccccgc gcgttggccg attcattaat gcagctggca cgacaggttt cccgactgga
     4321 aagcgggcag tgagcgcaac gcaattaatg tgagttagct cactcattag gcaccccagg
     4381 ctttacactt tatgcttccg gctcgtatgt tgtgtggaat tgtgagcgga taacaatttc
     4441 acacaggaaa cagctatgac catgattacg ccaagctcta gctagaggtc gacggtatac
     4501 agacatgata agatacattg atgagtttgg acaaaccaca actagaatgc agtgaaaaaa
     4561 atgctttatt tgtgaaattt gtgatgctat tgctttattt gtaaccatta taagctgcaa
     4621 taaacaagtt ggggtgggcg aagaactcca gcatgagatc cccgcgctgg aggatcatcc
     4681 agccggcgtc ccggaaaacg attccgaagc ccaacctttc atagaaggcg gcggtggaat
     4741 cgaaatctcg tagcacgtgt cagtcctgct cctcggccac gaagtgcacg cagttgccgg
     4801 ccgggtcgcg cagggcgaac tcccgccccc acggctgctc gccgatctcg gtcatggccg
     4861 gcccggaggc gtcccggaag ttcgtggaca cgacctccga ccactcggcg tacagctcgt
     4921 ccaggccgcg cacccacacc caggccaggg tgttgtccgg caccacctgg tcctggaccg
     4981 cgctgatgaa cagggtcacg tcgtcccgga ccacaccggc gaagtcgtcc tccacgaagt
     5041 cccgggagaa cccgagccgg tcggtccaga actcgaccgc tccggcgacg tcgcgcgcgg
     5101 tgagcaccgg aacggcactg gtcaacttgg ccatggttta gttcctcacc ttgtcgtatt
     5161 atactatgcc gatatactat gccgatgatt aattgtcaac acgtgctgat cagatccgaa
     5221 aatggatata caagctcccg ggagcttttt gcaaaagcct aggcctccaa aaaagcctcc
     5281 tcactacttc tggaatagct cagaggcaga ggcggcctcg gcctctgcat aaataaaaaa
     5341 aattagtcag ccatggggcg gagaatgggc ggaactgggc ggagttaggg gcgggatggg
     5401 cggagttagg ggcgggacta tggttgctga ctaattgaga tgcatgcttt gcatacttct
     5461 gcctgctggg gagcctgggg actttccaca cctggttgct gactaattga gatgcatgct
     5521 ttgcatactt ctgcctgctg gggagcctgg ggactttcca caccctaact gacacacatt
     5581 ccacagaatt aattcgcgtt aaatttttgt taaatcagct cattttttaa ccaataggcc
     5641 gaaatcggca aaatccctta taaatcaaaa gaatagaccg agatagggtt gagtgttgtt
     5701 ccagtttgga acaagagtcc actattaaag aacgtggact ccaacgtcaa agggcgaaaa
     5761 accgtctatc agggcgatgg cccactacgt gaaccatcac cctaatcaag ttttttgggg
     5821 tcgaggtgcc gtaaagcact aaatcggaac cctaaaggga gcccccgatt tagagcttga
     5881 cggggaaagc cggcgaacgt ggcgagaaag gaagggaaga aagcgaaagg agcgggcgct
     5941 agggcgctgg caagtgtagc ggtcacgctg cgcgtaacca ccacacccgc cgcgcttaat
     6001 gcgccgctac agggcgcgtg gggatacccc ctagagcccc agctggttct ttccgcctca
     6061 gaagccatag agcccaccgc atccccagca tgcctgctat tgtcttccca atcctccccc
     6121 ttgctgtcct gccccacccc accccccaga atagaatgac acctactcag acaatgcgat
     6181 gcaatttcct cattttatta ggaaaggaca gtgggagtgg caccttccag ggtcaaggaa
     6241 ggcacggggg aggggcaaac aacagatggc tggcaactag aaggcacagt cgaggctgat
     6301 cagcgggttt aaacgggccc tgctagagat tttccacact gactaaaagg gtctgaggga
     6361 tctctagtta ccagagtcac acaacagacg ggcacacact acttgaagca ctcaaggcaa
     6421 gctttattga ggcttaagca gtgggttccc tagttagcca gagagctccc aggctcagat
     6481 ctggtctaac cagagagacc cagtacaagc aaaaagcaga tcttgtcttc gttgggagtg
     6541 aattagccct tccagtcccc ccttttcttt taaaaagtgg ctaagatcta cagctgcctt
     6601 gtaagtcatt ggtcttaaag tcgacgcggg gaggcggccc aaagggagat ccgactcgtc
     6661 tgagggcgaa ggcgaagacg cggaagaggc cgcagagccg gcagcaggcc gcgggaagga
     6721 aggtccgctg gattgagggc cgaagggacg tagcagaagg acgtcccgcg cagaatccag
     6781 gtggcaacac aggcgagcag ccaaggaaag gacgatgatt tccccgacaa caccacggaa
     6841 ttgtcagtgc ccaacagccg agcccctgtc cagcagcggg caaggcaggc ggcgatgagt
     6901 tccgccgtgg caatagggag ggggaaagcg aaagtcccgg aaaggagctg acaggtggtg
     6961 gcaatgcccc aaccagtggg ggttgcgtca gcaaacacag tgcacaccac gccacgttgc
     7021 ctgacaacgg gccacaactc ctcataaaga gacagcaacc aggatttata caaggaggag
     7081 aaaatgaaag ccatacggga agcaatagca tgatacaaag gcattaaagc agcgtatcca
     7141 catagcgtaa aaggagcaac atagttaaga ataccagtca atctttcaca aattttgtaa
     7201 tccagaggtt gattgtcgac ttaacgcgtt caggcaccgg gcttgcgggt catgcaccag
     7261 gtgcgcggtc cttcgggcac ctcgacgtcg gcggtgacgg tgaagccgag ccgctcgtag
     7321 aaggggaggt tgcggggcgc ggaggtctcc aggaaggcgg gcaccccggc gcgctcggcc
     7381 gcctccactc cggggagcac gacggcgctg cccagaccct tgccctggtg gtcgggcgag
     7441 actccgacgg tggccaggaa ccacgcgggc tccttgggcc ggtgcggcgc caggaggcct
     7501 tccatctgtt gctgcgcggc cagccgggaa ccgctcaact cggccatgcg cgggccgatc
     7561 tcggcgaaca ccgcccccgc ttcgacgctc tccggcgtgg tccagaccgc caccgcggcg
     7621 ccgtcgtccg cgacccacac cttgccgatg tcgagcccga cgcgcgtgag gaagagttct
     7681 tgcagctcgg tgacccgctc gatgtggcgg tccggatcga cggtgtggcg cgtggcgggg
     7741 tagtcggcga acgcggcggc gagggtgcgt acggccctgg ggacgtcgtc gcgggtggcg
     7801 aggcgcaccg tgggcttgta ctcggtcggt ccaggattct cttcgacatc tccggcttgt
     7861 ttcagcagag agaagtttgt tgcgccggat cccttatcgt catcgtcttt gtaatctttc
     7921 ttcttcttag cctgtccagc cttctttgtg gcggcaggtc gcttgtcgcc tcccagctga
     7981 gacaggtcga tccgtgtctc gtacaggccg gtgatgctct ggtggatcag ggtggcgtcc
     8041 agcacctctt tggtgctggt gtacctcttc cggtcgatgg tggtgtcaaa gtacttgaag
     8101 gcggcagggg ctcccagatt ggtcagggta aacaggtgga tgatattctc ggcctgctct
     8161 ctgatgggct tatcccggtg cttgttgtag gcggacagca ctttgtccag attagcgtcg
     8221 gccaggatca ctctcttgga gaactcgctg atctgctcga tgatctcgtc caggtagtgc
     8281 ttgtgctgtt ccacaaacag ctgtttctgc tcattatcct cgggggagcc cttcagcttc
     8341 tcatagtggc tggccaggta caggaagttc acatatttgg agggcagggc cagttcgttt
     8401 cccttctgca gttcgccggc agaggccagc attctcttcc ggccgttttc cagctcgaac
     8461 agggagtact taggcagctt gatgatcagg tcctttttca cttctttgta gcccttggct
     8521 tccagaaagt cgatgggatt cttctcgaag ctgcttcttt ccatgatggt gatccccagc
     8581 agctctttca cactcttcag tttcttggac ttgccctttt ccactttggc caccaccagc
     8641 acagaatagg ccacggtggg gctgtcgaag ccgccgtact tcttagggtc ccagtccttc
     8701 tttctggcga tcagcttatc gctgttcctc ttgggcagga tagactcttt gctgaagccg
     8761 cctgtctgca cctcggtctt tttcacgata ttcacttggg gcatgctcag cactttccgc
     8821 acggtggcaa aatcccggcc cttatcccac acgatctccc cggtttcgcc gtttgtctcg
     8881 atcagaggcc gcttccggat ctcgccgttg gccagggtaa tctcggtctt gaaaaagttc
     8941 atgatgttgc tgtagaagaa gtacttggcg gtagccttgc cgatttcctg ctcgctcttg
     9001 gcgatcatct tccgcacgtc gtacaccttg tagtcgccgt acacgaactc gctttccagc
     9061 ttagggtact ttttgatcag ggcggttccc acgacggcgt tcaggtaggc gtcgtgggcg
     9121 tggtggtagt tgttgatctc gcgcactttg taaaactgga aatccttccg gaaatcggac
     9181 accagcttgg acttcagggt gatcactttc acttcccgga tcagcttgtc attctcgtcg
     9241 tacttagtgt tcatccggga gtccaggatc tgtgccacgt gctttgtgat ctgccgggtt
     9301 tccaccagct gtctcttgat gaagccggcc ttatccagtt cgctcaggcc gcctctctcg
     9361 gccttggtca gattgtcgaa ctttctctgg gtaatcagct tggcgttcag cagctgccgc
     9421 cagtagttct tcatcttctt cacgacctct tcggagggca cgttgtcgct cttgccccgg
     9481 ttcttgtcgc ttctggtcag caccttgttg tcgatggagt cgtccttcag aaagctctga
     9541 ggcacgatat ggtccacatc gtagtcggac agccggttga tgtccagttc ctggtccacg
     9601 tacatatccc gcccattctg caggtagtac aggtacagct tctcgttctg cagctgggtg
     9661 ttttccacgg ggtgttcttt caggatctgg ctgcccagct ctttgatgcc ctcttcgatc
     9721 cgcttcattc tctcgcggct gttcttctgt cccttctggg tggtctggtt ctctctggcc
     9781 atttcgatca cgatgttctc gggcttgtgc cggcccatca ctttcacgag ctcgtccacc
     9841 accttcactg tctgcaggat gcccttctta atggcggggc tgccggccag attggcaatg
     9901 tgctcgtgca ggctatcgcc ctggccggac acctgggctt tctggatgtc ctctttaaag
     9961 gtcaggctgt cgtcgtggat cagctgcatg aagtttctgt tggcgaagcc gtcggacttc
    10021 aggaaatcca ggattgtctt gccggactgc ttgtcccgga tgccgttgat cagcttccgg
    10081 ctcagcctgc cccagccggt gtatctccgc cgcttcagct gcttcatcac tttgtcgtcg
    10141 aacaggtggg cataggtttt cagccgttcc tcgatcatct ctctgtcctc aaacagtgtc
    10201 agggtcagca cgatatcttc cagaatgtcc tcgttttcct cattgtccag gaagtccttg
    10261 tccttgataa ttttcagcag atcgtggtat gtgcccaggg aggcgttgaa ccgatcttcc
    10321 acgccggaga tttccacgga gtcgaagcac tcgattttct tgaagtagtc ctctttcagc
    10381 tgcttcacgg tcactttccg gttggtcttg aacagcaggt ccacgatggc ctttttctgc
    10441 tcgccgctca ggaaggcggg ctttctcatt ccctcggtca cgtatttcac tttggtcagc
    10501 tcgttataca cggtgaagta ctcgtacagc aggctgtgct tgggcagcac cttctcgttg
    10561 ggcaggttct tatcgaagtt ggtcatccgc tcgatgaagc tctgggcgga agcgcccttg
    10621 tccaccactt cctcgaagtt ccagggggtg atggtttcct cgctctttct ggtcatccag
    10681 gcgaatctgc tgtttcccct ggccagaggg cccacgtagt aggggatgcg gaaggtcagg
    10741 atcttctcga tcttttcccg gttgtccttc aggaatgggt aaaaatcttc ctgccgccgc
    10801 agaatggcgt gcagctctcc caggtggatc tggtggggga tgctgccgtt gtcgaaggtc
    10861 cgctgcttcc gcagcaggtc ctctctgttc agcttcacga gcagttcctc ggtgccgtcc
    10921 atcttttcca ggatgggctt gatgaacttg tagaactctt cctggctggc tccgccgtca
    10981 atgtagccgg cgtagccgtt cttgctctgg tcgaagaaaa tctctttgta cttctcaggc
    11041 agctgctgcc gcacgagagc tttcagcagg gtcaggtcct ggtggtgctc gtcgtatctc
    11101 ttgatcatag aggcgctcag gggggccttg gtgatctcgg tgttcactct caggatgtcg
    11161 ctcagcagga tggcgtcgga caggttcttg gcggccagaa acaggtcggc gtactggtcg
    11221 ccgatctggg ccagcaggtt gtccaggtcg tcgtcgtagg tgtccttgct cagctgcagt
    11281 ttggcatcct cggccaggtc gaagttgctc ttgaagttgg gggtcaggcc caggctcagg
    11341 gcaatcaggt ttccgaacag gccattcttc ttctcgccgg gcagctgggc gatcagattt
    11401 tccagccgtc tgctcttgct cagtctggca gacaggatgg ccttggcgtc cacgccgctg
    11461 gcgttgatgg ggttttcctc gaacagctgg ttgtaggtct gcaccagctg gatgaacagc
    11521 ttgtccacgt cgctgttgtc ggggttcagg tcgccctcga tcaggaagtg gccccggaac
    11581 ttgatcatgt gggccagggc cagatagatc agccgcaggt cggccttgtc ggtgctgtcc
    11641 accagtttct ttctcaggtg gtagatggtg gggtacttct cgtggtaggc cacctcgtcc
    11701 acgatgttgc cgaagatggg gtgccgctcg tgcttcttat cctcttccac caggaaggac
    11761 tcttccagtc tgtggaagaa gctgtcgtcc accttggcca tctcgttgct gaagatctct
    11821 tgcagatagc agatccggtt cttccgtctg gtgtatcttc ttctggcggt tctcttcagc
    11881 cgggtggcct cggctgtttc gccgctgtcg aacagcaggg ctccgatcag gttcttcttg
    11941 atgctgtgcc ggtcggtgtt gcccagcacc ttgaatttct tgctgggcac cttgtactcg
    12001 tcggtgatca cggcccagcc cacagagttg gtgccgatgt ccaggccgat gctgtacttc
    12061 ttgtccatgg tggcagcgct ctagaaccgg tcctgtgttc tggcggcaaa cccgttgcga
    12121 aaaagaacgt tcacggcgac tactgcactt atatacggtt ctcccccacc ctcgggaaaa
    12181 aggcggagcc agtacacgac atcactttcc cagtttaccc cgcgccacct tctctaggca
    12241 ccggatcaat tgccgacccc tccccccaac ttctcgggga ctgtgggcga tgtgcgctct
    12301 gcccactgac gggcaccgga gccaattccc actcctttca agacctagct agcgaattca
    12361 aaaaagcacc gactcggtgc cactttttca agttgataac ggactagcct tattttaact
    12421 tgctatttct agctctaaaa cggtgttgag gtttccccgc tcggtgtttc gtcctttcca
    12481 caagatatat aaagccaaga aatcgaaata ctttcaagtt acggtaagca tatgatagtc
    12541 cattttaaaa cataatttta aaactgcaaa ctacccaaga aattattact ttctacgtca
    12601 cgtattttgt actaatatct ttgtgtttac agtcaaatta attctaatta tctctctaac
    12661 agccttgtat cgtatatgca aatatgaagg aatcatggga aataggccct cggtacctta
    12721 attaaccaaa ctggatctct gctgtccctg taataaaccc gaaaattttg aatttttgta
    12781 atttgttttt gtaattcttt agtttgtatg tctgttgcta ttatgtctac tattctttcc
    12841 cctgcactgt accccccaat cccccctttt cttttaaaat tgtggatgaa tactgccatt
    12901 tgtctgcaga attggcgcac gcagtgccga tccgttcact aatcgaatgg atctgtctct
    12961 gtctctctct ccaccttctt cttctattcc ttcgggcctg tcgggtcccc tcg
//
