LOCUS       sequence-364861-       13034 bp    DNA     circular SYN 15-DEC-2025
DEFINITION  knock out ACSL4 in mammalian cells.
ACCESSION   .
VERSION     .
KEYWORDS    .
SOURCE      synthetic DNA construct
  ORGANISM  synthetic DNA construct
REFERENCE   1  (bases 1 to 13034)
  AUTHORS   Mao C, Liu X, Zhang Y, Lei G, Yan Y, Lee H, Koppula P, Wu S, Zhuang 
            L, Fang B, Poyurovsky MV, Olszewski K, Gan B
  TITLE     DHODH-mediated ferroptosis defence is a targetable vulnerability in 
            cancer.
  JOURNAL   Nature. 2021 May;593(7860):586-590. doi: 10.1038/s41586-021-03539-7.
            Epub 2021 May 12.
  PUBMED    33981038
REFERENCE   2  (bases 1 to 13034)
  AUTHORS   .
  TITLE     Direct Submission
  JOURNAL   Exported Dec 15, 2025 from SnapGene Server 8.0.1
            https://www.snapgene.com
COMMENT     SGRef: number: 1; type: "Journal Article"; doi: 
            "10.1038/s41586-021-03539-7"; journalName: "Nature"; date: 
            "2021-05"; volume: "593"; issue: "7860"; pages: "586-590"
COMMENT     Sequence Label: lentiCRISPR v2-sgACSL4
FEATURES             Location/Qualifiers
     source          1..13034
                     /mol_type="other DNA"
                     /organism="synthetic DNA construct"
     primer_bind     11..32
                     /label=F1ori-F
                     /note="F1 origin, forward primer"
     primer_bind     complement(151..171)
                     /label=pBABE 3'
                     /note="SV40 enhancer, reverse primer for pBABE vectors"
     promoter        156..485
                     /label=SV40 promoter
                     /note="SV40 enhancer and early promoter"
     rep_origin      336..471
                     /label=SV40 ori
                     /note="SV40 origin of replication"
     primer_bind     398..417
                     /label=SV40pro-F
                     /note="SV40 promoter/origin, forward primer"
     promoter        533..580
                     /label=EM7 promoter
                     /note="synthetic bacterial promoter "
     CDS             599..973
                     /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    1103..1224
                     /label=SV40 poly(A) signal
                     /note="SV40 polyadenylation signal"
     primer_bind     complement(1140..1159)
                     /label=SV40pA-R
                     /note="SV40 polyA, reverse primer"
     primer_bind     1194..1213
                     /label=EBV-rev
                     /note="SV40 polyA terminator, reverse primer"
     primer_bind     complement(1273..1289)
                     /label=M13 rev
                     /note="common sequencing primer, one of multiple similar 
                     variants"
     primer_bind     complement(1273..1289)
                     /label=M13 Reverse
                     /note="In lacZ gene. Also called M13-rev"
     primer_bind     complement(1286..1308)
                     /label=M13/pUC Reverse
                     /note="In lacZ gene"
     protein_bind    1297..1313
                     /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(1321..1327,1328..1345,1346..1351))
                     /label=lac promoter
                     /note="promoter for the E. coli lac operon"
     protein_bind    1366..1387
                     /label=CAP binding site
                     /bound_moiety="E. coli catabolite activator protein"
                     /note="CAP binding activates transcription in the presence 
                     of cAMP."
     primer_bind     complement(1504..1521)
                     /label=L4440
                     /note="L4440 vector, forward primer"
     rep_origin      complement(1675..2263)
                     /direction=LEFT
                     /label=ori
                     /note="high-copy-number ColE1/pMB1/pBR322/pUC origin of 
                     replication"
     primer_bind     complement(1755..1774)
                     /label=pBR322ori-F
                     /note="pBR322 origin, forward primer"
     CDS             complement(join(2434..3225,3226..3294))
                     /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     3057..3076
                     /label=Amp-R
                     /note="Ampicillin resistance gene, reverse primer"
     promoter        complement(3295..3399)
                     /gene="bla"
                     /label=AmpR promoter
     primer_bind     complement(3474..3493)
                     /label=pRS-marker
                     /note="pRS vectors, use to sequence yeast selectable 
                     marker"
     enhancer        3665..4044
                     /label=CMV enhancer
                     /note="human cytomegalovirus immediate early enhancer"
     promoter        4046..4244
                     /label=CMV promoter
                     /note="human cytomegalovirus (CMV) immediate early 
                     promoter"
     primer_bind     4199..4219
                     /label=CMV-F
                     /note="Human CMV immediate early promoter, forward primer"
     LTR             4262..4442
                     /label=5' LTR (truncated)
                     /note="truncated 5' long terminal repeat (LTR) from HIV-1"
     misc_feature    4489..4614
                     /label=HIV-1 Psi
                     /note="packaging signal of human immunodeficiency virus 
                     type 1"
     misc_feature    5107..5340
                     /label=RRE
                     /note="The Rev response element (RRE) of HIV-1 allows for 
                     Rev-dependent mRNA export from the nucleus to the 
                     cytoplasm."
     CDS             5525..5569
                     /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    5867..5984
                     /label=cPPT/CTS
                     /note="central polypurine tract and central termination 
                     sequence of HIV-1"
     promoter        6035..6283
                     /label=U6 promoter
                     /note="RNA polymerase III promoter for human U6 snRNA 
                     (Domitrovich & Kunkel, 2003)"
     primer_bind     6035..6055
                     /label=hU6-F
                     /note="Human U6 promoter, forward primer"
     primer_bind     6206..6225
                     /label=LKO.1 5'
                     /note="Human U6 promoter, forward primer"
     misc_RNA        6326..6401
                     /label=gRNA scaffold
                     /note="guide RNA scaffold for the Streptococcus pyogenes 
                     CRISPR/Cas9 system"
     promoter        6463..6674
                     /label=EF-1-alpha core promoter
                     /note="core promoter for human elongation factor 
                     EF-1-alpha"
     regulatory      6693..6702
                     /label=Kozak sequence
                     /note="vertebrate consensus sequence for strong initiation 
                     of translation (Kozak, 1987)"
                     /regulatory_class="other"
     CDS             6699..10802
                     /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             6699..10802
                     /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             10803..10850
                     /codon_start=1
                     /product="bipartite nuclear localization signal from 
                     nucleoplasmin"
                     /label=nucleoplasmin NLS
                     /translation="KRPAATKKAGQAKKKK"
     CDS             10851..10874
                     /codon_start=1
                     /product="FLAG(R) epitope tag, followed by an enterokinase 
                     cleavage site"
                     /label=FLAG
                     /translation="DYKDDDDK"
     CDS             10884..10940
                     /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             10941..11537
                     /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     11434..11454
                     /label=Puro-F
                     /note="Puromycin resistance gene, forward primer"
     misc_feature    11553..12141
                     /label=WPRE
                     /note="woodchuck hepatitis virus posttranscriptional 
                     regulatory element"
     primer_bind     complement(11606..11626)
                     /label=WPRE-R
                     /note="WPRE, reverse primer"
     CDS             complement(12024..12035)
                     /codon_start=1
                     /product="Factor Xa recognition and cleavage site"
                     /label=Factor Xa site
                     /translation="IEGR"
     LTR             12213..12446
                     /label=3' LTR (Delta-U3)
                     /note="self-inactivating 3' long terminal repeat (LTR) from
                     HIV-1"
     primer_bind     complement(12472..12489)
                     /label=BGH-rev
                     /note="Bovine growth hormone terminator, reverse primer. 
                     Also called BGH reverse"
     polyA_signal    12478..12702
                     /label=bGH poly(A) signal
                     /note="bovine growth hormone polyadenylation signal"
     rep_origin      join(12748..13034,1..142)
                     /direction=RIGHT
                     /label=f1 ori
                     /note="f1 bacteriophage origin of replication; arrow 
                     indicates direction of (+) strand synthesis"
     primer_bind     complement(12835..12854)
                     /label=F1ori-R
                     /note="F1 origin, reverse primer"
ORIGIN
        1 ttctttaata gtggactctt gttccaaact ggaacaacac tcaaccctat ctcggtctat
       61 tcttttgatt tataagggat tttgccgatt tcggcctatt ggttaaaaaa tgagctgatt
      121 taacaaaaat ttaacgcgaa ttaattctgt ggaatgtgtg tcagttaggg tgtggaaagt
      181 ccccaggctc cccagcaggc agaagtatgc aaagcatgca tctcaattag tcagcaacca
      241 ggtgtggaaa gtccccaggc tccccagcag gcagaagtat gcaaagcatg catctcaatt
      301 agtcagcaac catagtcccg cccctaactc cgcccatccc gcccctaact ccgcccagtt
      361 ccgcccattc tccgccccat ggctgactaa ttttttttat ttatgcagag gccgaggccg
      421 cctctgcctc tgagctattc cagaagtagt gaggaggctt ttttggaggc ctaggctttt
      481 gcaaaaagct cccgggagct tgtatatcca ttttcggatc tgatcagcac gtgttgacaa
      541 ttaatcatcg gcatagtata tcggcatagt ataatacgac aaggtgagga actaaaccat
      601 ggccaagttg accagtgccg ttccggtgct caccgcgcgc gacgtcgccg gagcggtcga
      661 gttctggacc gaccggctcg ggttctcccg ggacttcgtg gaggacgact tcgccggtgt
      721 ggtccgggac gacgtgaccc tgttcatcag cgcggtccag gaccaggtgg tgccggacaa
      781 caccctggcc tgggtgtggg tgcgcggcct ggacgagctg tacgccgagt ggtcggaggt
      841 cgtgtccacg aacttccggg acgcctccgg gccggccatg accgagatcg gcgagcagcc
      901 gtgggggcgg gagttcgccc tgcgcgaccc ggccggcaac tgcgtgcact tcgtggccga
      961 ggagcaggac tgacacgtgc tacgagattt cgattccacc gccgccttct atgaaaggtt
     1021 gggcttcgga atcgttttcc gggacgccgg ctggatgatc ctccagcgcg gggatctcat
     1081 gctggagttc ttcgcccacc ccaacttgtt tattgcagct tataatggtt acaaataaag
     1141 caatagcatc acaaatttca caaataaagc atttttttca ctgcattcta gttgtggttt
     1201 gtccaaactc atcaatgtat cttatcatgt ctgtataccg tcgacctcta gctagagctt
     1261 ggcgtaatca tggtcatagc tgtttcctgt gtgaaattgt tatccgctca caattccaca
     1321 caacatacga gccggaagca taaagtgtaa agcctggggt gcctaatgag tgagctaact
     1381 cacattaatt gcgttgcgct cactgcccgc tttccagtcg ggaaacctgt cgtgccagct
     1441 gcattaatga atcggccaac gcgcggggag aggcggtttg cgtattgggc gctcttccgc
     1501 ttcctcgctc actgactcgc tgcgctcggt cgttcggctg cggcgagcgg tatcagctca
     1561 ctcaaaggcg gtaatacggt tatccacaga atcaggggat aacgcaggaa agaacatgtg
     1621 agcaaaaggc cagcaaaagg ccaggaaccg taaaaaggcc gcgttgctgg cgtttttcca
     1681 taggctccgc ccccctgacg agcatcacaa aaatcgacgc tcaagtcaga ggtggcgaaa
     1741 cccgacagga ctataaagat accaggcgtt tccccctgga agctccctcg tgcgctctcc
     1801 tgttccgacc ctgccgctta ccggatacct gtccgccttt ctcccttcgg gaagcgtggc
     1861 gctttctcat agctcacgct gtaggtatct cagttcggtg taggtcgttc gctccaagct
     1921 gggctgtgtg cacgaacccc ccgttcagcc cgaccgctgc gccttatccg gtaactatcg
     1981 tcttgagtcc aacccggtaa gacacgactt atcgccactg gcagcagcca ctggtaacag
     2041 gattagcaga gcgaggtatg taggcggtgc tacagagttc ttgaagtggt ggcctaacta
     2101 cggctacact agaagaacag tatttggtat ctgcgctctg ctgaagccag ttaccttcgg
     2161 aaaaagagtt ggtagctctt gatccggcaa acaaaccacc gctggtagcg gtggtttttt
     2221 tgtttgcaag cagcagatta cgcgcagaaa aaaaggatct caagaagatc ctttgatctt
     2281 ttctacgggg tctgacgctc agtggaacga aaactcacgt taagggattt tggtcatgag
     2341 attatcaaaa aggatcttca cctagatcct tttaaattaa aaatgaagtt ttaaatcaat
     2401 ctaaagtata tatgagtaaa cttggtctga cagttaccaa tgcttaatca gtgaggcacc
     2461 tatctcagcg atctgtctat ttcgttcatc catagttgcc tgactccccg tcgtgtagat
     2521 aactacgata cgggagggct taccatctgg ccccagtgct gcaatgatac cgcgagaccc
     2581 acgctcaccg gctccagatt tatcagcaat aaaccagcca gccggaaggg ccgagcgcag
     2641 aagtggtcct gcaactttat ccgcctccat ccagtctatt aattgttgcc gggaagctag
     2701 agtaagtagt tcgccagtta atagtttgcg caacgttgtt gccattgcta caggcatcgt
     2761 ggtgtcacgc tcgtcgtttg gtatggcttc attcagctcc ggttcccaac gatcaaggcg
     2821 agttacatga tcccccatgt tgtgcaaaaa agcggttagc tccttcggtc ctccgatcgt
     2881 tgtcagaagt aagttggccg cagtgttatc actcatggtt atggcagcac tgcataattc
     2941 tcttactgtc atgccatccg taagatgctt ttctgtgact ggtgagtact caaccaagtc
     3001 attctgagaa tagtgtatgc ggcgaccgag ttgctcttgc ccggcgtcaa tacgggataa
     3061 taccgcgcca catagcagaa ctttaaaagt gctcatcatt ggaaaacgtt cttcggggcg
     3121 aaaactctca aggatcttac cgctgttgag atccagttcg atgtaaccca ctcgtgcacc
     3181 caactgatct tcagcatctt ttactttcac cagcgtttct gggtgagcaa aaacaggaag
     3241 gcaaaatgcc gcaaaaaagg gaataagggc gacacggaaa tgttgaatac tcatactctt
     3301 cctttttcaa tattattgaa gcatttatca gggttattgt ctcatgagcg gatacatatt
     3361 tgaatgtatt tagaaaaata aacaaatagg ggttccgcgc acatttcccc gaaaagtgcc
     3421 acctgacgtc gacggatcgg gagatctccc gatcccctat ggtgcactct cagtacaatc
     3481 tgctctgatg ccgcatagtt aagccagtat ctgctccctg cttgtgtgtt ggaggtcgct
     3541 gagtagtgcg cgagcaaaat ttaagctaca acaaggcaag gcttgaccga caattgcatg
     3601 aagaatctgc ttagggttag gcgttttgcg ctgcttcgcg atgtacgggc cagatatacg
     3661 cgttgacatt gattattgac tagttattaa tagtaatcaa ttacggggtc attagttcat
     3721 agcccatata tggagttccg cgttacataa cttacggtaa atggcccgcc tggctgaccg
     3781 cccaacgacc cccgcccatt gacgtcaata atgacgtatg ttcccatagt aacgccaata
     3841 gggactttcc attgacgtca atgggtggag tatttacggt aaactgccca cttggcagta
     3901 catcaagtgt atcatatgcc aagtacgccc cctattgacg tcaatgacgg taaatggccc
     3961 gcctggcatt atgcccagta catgacctta tgggactttc ctacttggca gtacatctac
     4021 gtattagtca tcgctattac catggtgatg cggttttggc agtacatcaa tgggcgtgga
     4081 tagcggtttg actcacgggg atttccaagt ctccacccca ttgacgtcaa tgggagtttg
     4141 ttttggcacc aaaatcaacg ggactttcca aaatgtcgta acaactccgc cccattgacg
     4201 caaatgggcg gtaggcgtgt acggtgggag gtctatataa gcagcgcgtt ttgcctgtac
     4261 tgggtctctc tggttagacc agatctgagc ctgggagctc tctggctaac tagggaaccc
     4321 actgcttaag cctcaataaa gcttgccttg agtgcttcaa gtagtgtgtg cccgtctgtt
     4381 gtgtgactct ggtaactaga gatccctcag acccttttag tcagtgtgga aaatctctag
     4441 cagtggcgcc cgaacaggga cttgaaagcg aaagggaaac cagaggagct ctctcgacgc
     4501 aggactcggc ttgctgaagc gcgcacggca agaggcgagg ggcggcgact ggtgagtacg
     4561 ccaaaaattt tgactagcgg aggctagaag gagagagatg ggtgcgagag cgtcagtatt
     4621 aagcggggga gaattagatc gcgatgggaa aaaattcggt taaggccagg gggaaagaaa
     4681 aaatataaat taaaacatat agtatgggca agcagggagc tagaacgatt cgcagttaat
     4741 cctggcctgt tagaaacatc agaaggctgt agacaaatac tgggacagct acaaccatcc
     4801 cttcagacag gatcagaaga acttagatca ttatataata cagtagcaac cctctattgt
     4861 gtgcatcaaa ggatagagat aaaagacacc aaggaagctt tagacaagat agaggaagag
     4921 caaaacaaaa gtaagaccac cgcacagcaa gcggccgctg atcttcagac ctggaggagg
     4981 agatatgagg gacaattgga gaagtgaatt atataaatat aaagtagtaa aaattgaacc
     5041 attaggagta gcacccacca aggcaaagag aagagtggtg cagagagaaa aaagagcagt
     5101 gggaatagga gctttgttcc ttgggttctt gggagcagca ggaagcacta tgggcgcagc
     5161 gtcaatgacg ctgacggtac aggccagaca attattgtct ggtatagtgc agcagcagaa
     5221 caatttgctg agggctattg aggcgcaaca gcatctgttg caactcacag tctggggcat
     5281 caagcagctc caggcaagaa tcctggctgt ggaaagatac ctaaaggatc aacagctcct
     5341 ggggatttgg ggttgctctg gaaaactcat ttgcaccact gctgtgcctt ggaatgctag
     5401 ttggagtaat aaatctctgg aacagatttg gaatcacacg acctggatgg agtgggacag
     5461 agaaattaac aattacacaa gcttaataca ctccttaatt gaagaatcgc aaaaccagca
     5521 agaaaagaat gaacaagaat tattggaatt agataaatgg gcaagtttgt ggaattggtt
     5581 taacataaca aattggctgt ggtatataaa attattcata atgatagtag gaggcttggt
     5641 aggtttaaga atagtttttg ctgtactttc tatagtgaat agagttaggc agggatattc
     5701 accattatcg tttcagaccc acctcccaac cccgagggga cccgacaggc ccgaaggaat
     5761 agaagaagaa ggtggagaga gagacagaga cagatccatt cgattagtga acggatcggc
     5821 actgcgtgcg ccaattctgc agacaaatgg cagtattcat ccacaatttt aaaagaaaag
     5881 gggggattgg ggggtacagt gcaggggaaa gaatagtaga cataatagca acagacatac
     5941 aaactaaaga attacaaaaa caaattacaa aaattcaaaa ttttcgggtt tattacaggg
     6001 acagcagaga tccagtttgg ttaattaagg taccgagggc ctatttccca tgattccttc
     6061 atatttgcat atacgataca aggctgttag agagataatt agaattaatt tgactgtaaa
     6121 cacaaagata ttagtacaaa atacgtgacg tagaaagtaa taatttcttg ggtagtttgc
     6181 agttttaaaa ttatgtttta aaatggacta tcatatgctt accgtaactt gaaagtattt
     6241 cgatttcttg gctttatata tcttgtggaa aggacgaaac accgtgcaat catccattcg
     6301 gcccgtgcaa tcatccattc ggcccgtttt agagctagaa atagcaagtt aaaataaggc
     6361 tagtccgtta tcaacttgaa aaagtggcac cgagtcggtg cttttttgaa ttcgctagct
     6421 aggtcttgaa aggagtggga attggctccg gtgcccgtca gtgggcagag cgcacatcgc
     6481 ccacagtccc cgagaagttg gggggagggg tcggcaattg atccggtgcc tagagaaggt
     6541 ggcgcggggt aaactgggaa agtgatgtcg tgtactggct ccgccttttt cccgagggtg
     6601 ggggagaacc gtatataagt gcagtagtcg ccgtgaacgt tctttttcgc aacgggtttg
     6661 ccgccagaac acaggaccgg ttctagagcg ctgccaccat ggacaagaag tacagcatcg
     6721 gcctggacat cggcaccaac tctgtgggct gggccgtgat caccgacgag tacaaggtgc
     6781 ccagcaagaa attcaaggtg ctgggcaaca ccgaccggca cagcatcaag aagaacctga
     6841 tcggagccct gctgttcgac agcggcgaaa cagccgaggc cacccggctg aagagaaccg
     6901 ccagaagaag atacaccaga cggaagaacc ggatctgcta tctgcaagag atcttcagca
     6961 acgagatggc caaggtggac gacagcttct tccacagact ggaagagtcc ttcctggtgg
     7021 aagaggataa gaagcacgag cggcacccca tcttcggcaa catcgtggac gaggtggcct
     7081 accacgagaa gtaccccacc atctaccacc tgagaaagaa actggtggac agcaccgaca
     7141 aggccgacct gcggctgatc tatctggccc tggcccacat gatcaagttc cggggccact
     7201 tcctgatcga gggcgacctg aaccccgaca acagcgacgt ggacaagctg ttcatccagc
     7261 tggtgcagac ctacaaccag ctgttcgagg aaaaccccat caacgccagc ggcgtggacg
     7321 ccaaggccat cctgtctgcc agactgagca agagcagacg gctggaaaat ctgatcgccc
     7381 agctgcccgg cgagaagaag aatggcctgt tcggaaacct gattgccctg agcctgggcc
     7441 tgacccccaa cttcaagagc aacttcgacc tggccgagga tgccaaactg cagctgagca
     7501 aggacaccta cgacgacgac ctggacaacc tgctggccca gatcggcgac cagtacgccg
     7561 acctgtttct ggccgccaag aacctgtccg acgccatcct gctgagcgac atcctgagag
     7621 tgaacaccga gatcaccaag gcccccctga gcgcctctat gatcaagaga tacgacgagc
     7681 accaccagga cctgaccctg ctgaaagctc tcgtgcggca gcagctgcct gagaagtaca
     7741 aagagatttt cttcgaccag agcaagaacg gctacgccgg ctacattgac ggcggagcca
     7801 gccaggaaga gttctacaag ttcatcaagc ccatcctgga aaagatggac ggcaccgagg
     7861 aactgctcgt gaagctgaac agagaggacc tgctgcggaa gcagcggacc ttcgacaacg
     7921 gcagcatccc ccaccagatc cacctgggag agctgcacgc cattctgcgg cggcaggaag
     7981 atttttaccc attcctgaag gacaaccggg aaaagatcga gaagatcctg accttccgca
     8041 tcccctacta cgtgggccct ctggccaggg gaaacagcag attcgcctgg atgaccagaa
     8101 agagcgagga aaccatcacc ccctggaact tcgaggaagt ggtggacaag ggcgcttccg
     8161 cccagagctt catcgagcgg atgaccaact tcgataagaa cctgcccaac gagaaggtgc
     8221 tgcccaagca cagcctgctg tacgagtact tcaccgtgta taacgagctg accaaagtga
     8281 aatacgtgac cgagggaatg agaaagcccg ccttcctgag cggcgagcag aaaaaggcca
     8341 tcgtggacct gctgttcaag accaaccgga aagtgaccgt gaagcagctg aaagaggact
     8401 acttcaagaa aatcgagtgc ttcgactccg tggaaatctc cggcgtggaa gatcggttca
     8461 acgcctccct gggcacatac cacgatctgc tgaaaattat caaggacaag gacttcctgg
     8521 acaatgagga aaacgaggac attctggaag atatcgtgct gaccctgaca ctgtttgagg
     8581 acagagagat gatcgaggaa cggctgaaaa cctatgccca cctgttcgac gacaaagtga
     8641 tgaagcagct gaagcggcgg agatacaccg gctggggcag gctgagccgg aagctgatca
     8701 acggcatccg ggacaagcag tccggcaaga caatcctgga tttcctgaag tccgacggct
     8761 tcgccaacag aaacttcatg cagctgatcc acgacgacag cctgaccttt aaagaggaca
     8821 tccagaaagc ccaggtgtcc ggccagggcg atagcctgca cgagcacatt gccaatctgg
     8881 ccggcagccc cgccattaag aagggcatcc tgcagacagt gaaggtggtg gacgagctcg
     8941 tgaaagtgat gggccggcac aagcccgaga acatcgtgat cgaaatggcc agagagaacc
     9001 agaccaccca gaagggacag aagaacagcc gcgagagaat gaagcggatc gaagagggca
     9061 tcaaagagct gggcagccag atcctgaaag aacaccccgt ggaaaacacc cagctgcaga
     9121 acgagaagct gtacctgtac tacctgcaga atgggcggga tatgtacgtg gaccaggaac
     9181 tggacatcaa ccggctgtcc gactacgatg tggaccatat cgtgcctcag agctttctga
     9241 aggacgactc catcgacaac aaggtgctga ccagaagcga caagaaccgg ggcaagagcg
     9301 acaacgtgcc ctccgaagag gtcgtgaaga agatgaagaa ctactggcgg cagctgctga
     9361 acgccaagct gattacccag agaaagttcg acaatctgac caaggccgag agaggcggcc
     9421 tgagcgaact ggataaggcc ggcttcatca agagacagct ggtggaaacc cggcagatca
     9481 caaagcacgt ggcacagatc ctggactccc ggatgaacac taagtacgac gagaatgaca
     9541 agctgatccg ggaagtgaaa gtgatcaccc tgaagtccaa gctggtgtcc gatttccgga
     9601 aggatttcca gttttacaaa gtgcgcgaga tcaacaacta ccaccacgcc cacgacgcct
     9661 acctgaacgc cgtcgtggga accgccctga tcaaaaagta ccctaagctg gaaagcgagt
     9721 tcgtgtacgg cgactacaag gtgtacgacg tgcggaagat gatcgccaag agcgagcagg
     9781 aaatcggcaa ggctaccgcc aagtacttct tctacagcaa catcatgaac tttttcaaga
     9841 ccgagattac cctggccaac ggcgagatcc ggaagcggcc tctgatcgag acaaacggcg
     9901 aaaccgggga gatcgtgtgg gataagggcc gggattttgc caccgtgcgg aaagtgctga
     9961 gcatgcccca agtgaatatc gtgaaaaaga ccgaggtgca gacaggcggc ttcagcaaag
    10021 agtctatcct gcccaagagg aacagcgata agctgatcgc cagaaagaag gactgggacc
    10081 ctaagaagta cggcggcttc gacagcccca ccgtggccta ttctgtgctg gtggtggcca
    10141 aagtggaaaa gggcaagtcc aagaaactga agagtgtgaa agagctgctg gggatcacca
    10201 tcatggaaag aagcagcttc gagaagaatc ccatcgactt tctggaagcc aagggctaca
    10261 aagaagtgaa aaaggacctg atcatcaagc tgcctaagta ctccctgttc gagctggaaa
    10321 acggccggaa gagaatgctg gcctctgccg gcgaactgca gaagggaaac gaactggccc
    10381 tgccctccaa atatgtgaac ttcctgtacc tggccagcca ctatgagaag ctgaagggct
    10441 cccccgagga taatgagcag aaacagctgt ttgtggaaca gcacaagcac tacctggacg
    10501 agatcatcga gcagatcagc gagttctcca agagagtgat cctggccgac gctaatctgg
    10561 acaaagtgct gtccgcctac aacaagcacc gggataagcc catcagagag caggccgaga
    10621 atatcatcca cctgtttacc ctgaccaatc tgggagcccc tgccgccttc aagtactttg
    10681 acaccaccat cgaccggaag aggtacacca gcaccaaaga ggtgctggac gccaccctga
    10741 tccaccagag catcaccggc ctgtacgaga cacggatcga cctgtctcag ctgggaggcg
    10801 acaagcgacc tgccgccaca aagaaggctg gacaggctaa gaagaagaaa gattacaaag
    10861 acgatgacga taagggatcc ggcgcaacaa acttctctct gctgaaacaa gccggagatg
    10921 tcgaagagaa tcctggaccg accgagtaca agcccacggt gcgcctcgcc acccgcgacg
    10981 acgtccccag ggccgtacgc accctcgccg ccgcgttcgc cgactacccc gccacgcgcc
    11041 acaccgtcga tccggaccgc cacatcgagc gggtcaccga gctgcaagaa ctcttcctca
    11101 cgcgcgtcgg gctcgacatc ggcaaggtgt gggtcgcgga cgacggcgcc gcggtggcgg
    11161 tctggaccac gccggagagc gtcgaagcgg gggcggtgtt cgccgagatc ggcccgcgca
    11221 tggccgagtt gagcggttcc cggctggccg cgcagcaaca gatggaaggc ctcctggcgc
    11281 cgcaccggcc caaggagccc gcgtggttcc tggccaccgt cggagtctcg cccgaccacc
    11341 agggcaaggg tctgggcagc gccgtcgtgc tccccggagt ggaggcggcc gagcgcgccg
    11401 gggtgcccgc cttcctggag acctccgcgc cccgcaacct ccccttctac gagcggctcg
    11461 gcttcaccgt caccgccgac gtcgaggtgc ccgaaggacc gcgcacctgg tgcatgaccc
    11521 gcaagcccgg tgcctgaacg cgttaagtcg acaatcaacc tctggattac aaaatttgtg
    11581 aaagattgac tggtattctt aactatgttg ctccttttac gctatgtgga tacgctgctt
    11641 taatgccttt gtatcatgct attgcttccc gtatggcttt cattttctcc tccttgtata
    11701 aatcctggtt gctgtctctt tatgaggagt tgtggcccgt tgtcaggcaa cgtggcgtgg
    11761 tgtgcactgt gtttgctgac gcaaccccca ctggttgggg cattgccacc acctgtcagc
    11821 tcctttccgg gactttcgct ttccccctcc ctattgccac ggcggaactc atcgccgcct
    11881 gccttgcccg ctgctggaca ggggctcggc tgttgggcac tgacaattcc gtggtgttgt
    11941 cggggaaatc atcgtccttt ccttggctgc tcgcctgtgt tgccacctgg attctgcgcg
    12001 ggacgtcctt ctgctacgtc ccttcggccc tcaatccagc ggaccttcct tcccgcggcc
    12061 tgctgccggc tctgcggcct cttccgcgtc ttcgccttcg ccctcagacg agtcggatct
    12121 ccctttgggc cgcctccccg cgtcgacttt aagaccaatg acttacaagg cagctgtaga
    12181 tcttagccac tttttaaaag aaaagggggg actggaaggg ctaattcact cccaacgaag
    12241 acaagatctg ctttttgctt gtactgggtc tctctggtta gaccagatct gagcctggga
    12301 gctctctggc taactaggga acccactgct taagcctcaa taaagcttgc cttgagtgct
    12361 tcaagtagtg tgtgcccgtc tgttgtgtga ctctggtaac tagagatccc tcagaccctt
    12421 ttagtcagtg tggaaaatct ctagcagggc ccgtttaaac ccgctgatca gcctcgactg
    12481 tgccttctag ttgccagcca tctgttgttt gcccctcccc cgtgccttcc ttgaccctgg
    12541 aaggtgccac tcccactgtc ctttcctaat aaaatgagga aattgcatcg cattgtctga
    12601 gtaggtgtca ttctattctg gggggtgggg tggggcagga cagcaagggg gaggattggg
    12661 aagacaatag caggcatgct ggggatgcgg tgggctctat ggcttctgag gcggaaagaa
    12721 ccagctgggg ctctaggggg tatccccacg cgccctgtag cggcgcatta agcgcggcgg
    12781 gtgtggtggt tacgcgcagc gtgaccgcta cacttgccag cgccctagcg cccgctcctt
    12841 tcgctttctt cccttccttt ctcgccacgt tcgccggctt tccccgtcaa gctctaaatc
    12901 gggggctccc tttagggttc cgatttagtg ctttacggca cctcgacccc aaaaaacttg
    12961 attagggtga tggttcacgt agtgggccat cgccctgata gacggttttt cgccctttga
    13021 cgttggagtc cacg
//
