LOCUS       sequence-413628-       13013 bp    DNA     circular SYN 15-DEC-2025
DEFINITION  knock out BAK in mammalian cells.
ACCESSION   .
VERSION     .
KEYWORDS    .
SOURCE      synthetic DNA construct
  ORGANISM  synthetic DNA construct
REFERENCE   1  (bases 1 to 13013)
  AUTHORS   Liu X, Nie L, Zhang Y, Yan Y, Wang C, Colic M, Olszewski K, Horbath 
            A, Chen X, Lei G, Mao C, Wu S, Zhuang L, Poyurovsky MV, James You M,
            Hart T, Billadeau DD, Chen J, Gan B
  TITLE     Actin cytoskeleton vulnerability to disulfide stress mediates 
            disulfidptosis.
  JOURNAL   Nat Cell Biol. 2023 Mar;25(3):404-414. doi: 
            10.1038/s41556-023-01091-2. Epub 2023 Feb 6.
  PUBMED    36747082
REFERENCE   2  (bases 1 to 13013)
  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/s41556-023-01091-2"; journalName: "Nat Cell Biol"; date: 
            "2023-03"; volume: "25"; issue: "3"; pages: "404-414"
COMMENT     Sequence Label: lentiCRISPR v2-sgBAK-1
FEATURES             Location/Qualifiers
     source          1..13013
                     /mol_type="other DNA"
                     /organism="synthetic DNA construct"
     primer_bind     111..131
                     /label=CMV-F
                     /note="Human CMV immediate early promoter, forward primer"
     LTR             174..354
                     /label=5' LTR (truncated)
                     /note="truncated 5' long terminal repeat (LTR) from HIV-1"
     misc_feature    401..526
                     /label=HIV-1 Psi
                     /note="packaging signal of human immunodeficiency virus 
                     type 1"
     misc_feature    1019..1252
                     /label=RRE
                     /note="The Rev response element (RRE) of HIV-1 allows for 
                     Rev-dependent mRNA export from the nucleus to the 
                     cytoplasm."
     CDS             1437..1481
                     /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    1779..1896
                     /label=cPPT/CTS
                     /note="central polypurine tract and central termination 
                     sequence of HIV-1"
     promoter        1947..2195
                     /label=U6 promoter
                     /note="RNA polymerase III promoter for human U6 snRNA 
                     (Domitrovich & Kunkel, 2003)"
     primer_bind     1947..1967
                     /label=hU6-F
                     /note="Human U6 promoter, forward primer"
     primer_bind     2118..2137
                     /label=LKO.1 5'
                     /note="Human U6 promoter, forward primer"
     misc_RNA        2217..2292
                     /label=gRNA scaffold
                     /note="guide RNA scaffold for the Streptococcus pyogenes 
                     CRISPR/Cas9 system"
     promoter        2354..2565
                     /label=EF-1-alpha core promoter
                     /note="core promoter for human elongation factor 
                     EF-1-alpha"
     regulatory      2584..2593
                     /label=Kozak sequence
                     /note="vertebrate consensus sequence for strong initiation 
                     of translation (Kozak, 1987)"
                     /regulatory_class="other"
     CDS             2590..6693
                     /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             2590..6693
                     /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             6694..6741
                     /codon_start=1
                     /product="bipartite nuclear localization signal from 
                     nucleoplasmin"
                     /label=nucleoplasmin NLS
                     /translation="KRPAATKKAGQAKKKK"
     CDS             6742..6765
                     /codon_start=1
                     /product="FLAG(R) epitope tag, followed by an enterokinase 
                     cleavage site"
                     /label=FLAG
                     /translation="DYKDDDDK"
     CDS             6775..6831
                     /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             6832..7428
                     /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     7325..7345
                     /label=Puro-F
                     /note="Puromycin resistance gene, forward primer"
     misc_feature    7444..8032
                     /label=WPRE
                     /note="woodchuck hepatitis virus posttranscriptional 
                     regulatory element"
     primer_bind     complement(7497..7517)
                     /label=WPRE-R
                     /note="WPRE, reverse primer"
     CDS             complement(7915..7926)
                     /codon_start=1
                     /product="Factor Xa recognition and cleavage site"
                     /label=Factor Xa site
                     /translation="IEGR"
     LTR             8104..8337
                     /label=3' LTR (Delta-U3)
                     /note="self-inactivating 3' long terminal repeat (LTR) from
                     HIV-1"
     primer_bind     complement(8363..8380)
                     /label=BGH-rev
                     /note="Bovine growth hormone terminator, reverse primer. 
                     Also called BGH reverse"
     polyA_signal    8369..8593
                     /label=bGH poly(A) signal
                     /note="bovine growth hormone polyadenylation signal"
     rep_origin      8639..9067
                     /direction=RIGHT
                     /label=f1 ori
                     /note="f1 bacteriophage origin of replication; arrow 
                     indicates direction of (+) strand synthesis"
     primer_bind     complement(8726..8745)
                     /label=F1ori-R
                     /note="F1 origin, reverse primer"
     primer_bind     8936..8957
                     /label=F1ori-F
                     /note="F1 origin, forward primer"
     primer_bind     complement(9076..9096)
                     /label=pBABE 3'
                     /note="SV40 enhancer, reverse primer for pBABE vectors"
     promoter        9081..9410
                     /label=SV40 promoter
                     /note="SV40 enhancer and early promoter"
     rep_origin      9261..9396
                     /label=SV40 ori
                     /note="SV40 origin of replication"
     primer_bind     9323..9342
                     /label=SV40pro-F
                     /note="SV40 promoter/origin, forward primer"
     promoter        9458..9505
                     /label=EM7 promoter
                     /note="synthetic bacterial promoter "
     CDS             9524..9898
                     /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    10028..10149
                     /label=SV40 poly(A) signal
                     /note="SV40 polyadenylation signal"
     primer_bind     complement(10065..10084)
                     /label=SV40pA-R
                     /note="SV40 polyA, reverse primer"
     primer_bind     10119..10138
                     /label=EBV-rev
                     /note="SV40 polyA terminator, reverse primer"
     primer_bind     complement(10198..10214)
                     /label=M13 rev
                     /note="common sequencing primer, one of multiple similar 
                     variants"
     primer_bind     complement(10198..10214)
                     /label=M13 Reverse
                     /note="In lacZ gene. Also called M13-rev"
     primer_bind     complement(10211..10233)
                     /label=M13/pUC Reverse
                     /note="In lacZ gene"
     protein_bind    10222..10238
                     /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(10246..10252,10253..10270,10271..10276))
                     /label=lac promoter
                     /note="promoter for the E. coli lac operon"
     protein_bind    10291..10312
                     /label=CAP binding site
                     /bound_moiety="E. coli catabolite activator protein"
                     /note="CAP binding activates transcription in the presence 
                     of cAMP."
     primer_bind     complement(10429..10446)
                     /label=L4440
                     /note="L4440 vector, forward primer"
     rep_origin      complement(10600..11188)
                     /direction=LEFT
                     /label=ori
                     /note="high-copy-number ColE1/pMB1/pBR322/pUC origin of 
                     replication"
     primer_bind     complement(10680..10699)
                     /label=pBR322ori-F
                     /note="pBR322 origin, forward primer"
     CDS             complement(join(11359..12150,12151..12219))
                     /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     11982..12001
                     /label=Amp-R
                     /note="Ampicillin resistance gene, reverse primer"
     promoter        complement(12220..12324)
                     /gene="bla"
                     /label=AmpR promoter
     primer_bind     complement(12399..12418)
                     /label=pRS-marker
                     /note="pRS vectors, use to sequence yeast selectable 
                     marker"
     enhancer        12590..12969
                     /label=CMV enhancer
                     /note="human cytomegalovirus immediate early enhancer"
     promoter        join(12971..13013,1..156)
                     /label=CMV promoter
                     /note="human cytomegalovirus (CMV) immediate early 
                     promoter"
ORIGIN
        1 tgactcacgg ggatttccaa gtctccaccc cattgacgtc aatgggagtt tgttttggca
       61 ccaaaatcaa cgggactttc caaaatgtcg taacaactcc gccccattga cgcaaatggg
      121 cggtaggcgt gtacggtggg aggtctatat aagcagcgcg ttttgcctgt actgggtctc
      181 tctggttaga ccagatctga gcctgggagc tctctggcta actagggaac ccactgctta
      241 agcctcaata aagcttgcct tgagtgcttc aagtagtgtg tgcccgtctg ttgtgtgact
      301 ctggtaacta gagatccctc agaccctttt agtcagtgtg gaaaatctct agcagtggcg
      361 cccgaacagg gacttgaaag cgaaagggaa accagaggag ctctctcgac gcaggactcg
      421 gcttgctgaa gcgcgcacgg caagaggcga ggggcggcga ctggtgagta cgccaaaaat
      481 tttgactagc ggaggctaga aggagagaga tgggtgcgag agcgtcagta ttaagcgggg
      541 gagaattaga tcgcgatggg aaaaaattcg gttaaggcca gggggaaaga aaaaatataa
      601 attaaaacat atagtatggg caagcaggga gctagaacga ttcgcagtta atcctggcct
      661 gttagaaaca tcagaaggct gtagacaaat actgggacag ctacaaccat cccttcagac
      721 aggatcagaa gaacttagat cattatataa tacagtagca accctctatt gtgtgcatca
      781 aaggatagag ataaaagaca ccaaggaagc tttagacaag atagaggaag agcaaaacaa
      841 aagtaagacc accgcacagc aagcggccgc tgatcttcag acctggagga ggagatatga
      901 gggacaattg gagaagtgaa ttatataaat ataaagtagt aaaaattgaa ccattaggag
      961 tagcacccac caaggcaaag agaagagtgg tgcagagaga aaaaagagca gtgggaatag
     1021 gagctttgtt ccttgggttc ttgggagcag caggaagcac tatgggcgca gcgtcaatga
     1081 cgctgacggt acaggccaga caattattgt ctggtatagt gcagcagcag aacaatttgc
     1141 tgagggctat tgaggcgcaa cagcatctgt tgcaactcac agtctggggc atcaagcagc
     1201 tccaggcaag aatcctggct gtggaaagat acctaaagga tcaacagctc ctggggattt
     1261 ggggttgctc tggaaaactc atttgcacca ctgctgtgcc ttggaatgct agttggagta
     1321 ataaatctct ggaacagatt tggaatcaca cgacctggat ggagtgggac agagaaatta
     1381 acaattacac aagcttaata cactccttaa ttgaagaatc gcaaaaccag caagaaaaga
     1441 atgaacaaga attattggaa ttagataaat gggcaagttt gtggaattgg tttaacataa
     1501 caaattggct gtggtatata aaattattca taatgatagt aggaggcttg gtaggtttaa
     1561 gaatagtttt tgctgtactt tctatagtga atagagttag gcagggatat tcaccattat
     1621 cgtttcagac ccacctccca accccgaggg gacccgacag gcccgaagga atagaagaag
     1681 aaggtggaga gagagacaga gacagatcca ttcgattagt gaacggatcg gcactgcgtg
     1741 cgccaattct gcagacaaat ggcagtattc atccacaatt ttaaaagaaa aggggggatt
     1801 ggggggtaca gtgcagggga aagaatagta gacataatag caacagacat acaaactaaa
     1861 gaattacaaa aacaaattac aaaaattcaa aattttcggg tttattacag ggacagcaga
     1921 gatccagttt ggttaattaa ggtaccgagg gcctatttcc catgattcct tcatatttgc
     1981 atatacgata caaggctgtt agagagataa ttagaattaa tttgactgta aacacaaaga
     2041 tattagtaca aaatacgtga cgtagaaagt aataatttct tgggtagttt gcagttttaa
     2101 aattatgttt taaaatggac tatcatatgc ttaccgtaac ttgaaagtat ttcgatttct
     2161 tggctttata tatcttgtgg aaaggacgaa acaccggcag gtagcccagg acacaggttt
     2221 tagagctaga aatagcaagt taaaataagg ctagtccgtt atcaacttga aaaagtggca
     2281 ccgagtcggt gcttttttga attcgctagc taggtcttga aaggagtggg aattggctcc
     2341 ggtgcccgtc agtgggcaga gcgcacatcg cccacagtcc ccgagaagtt ggggggaggg
     2401 gtcggcaatt gatccggtgc ctagagaagg tggcgcgggg taaactggga aagtgatgtc
     2461 gtgtactggc tccgcctttt tcccgagggt gggggagaac cgtatataag tgcagtagtc
     2521 gccgtgaacg ttctttttcg caacgggttt gccgccagaa cacaggaccg gttctagagc
     2581 gctgccacca tggacaagaa gtacagcatc ggcctggaca tcggcaccaa ctctgtgggc
     2641 tgggccgtga tcaccgacga gtacaaggtg cccagcaaga aattcaaggt gctgggcaac
     2701 accgaccggc acagcatcaa gaagaacctg atcggagccc tgctgttcga cagcggcgaa
     2761 acagccgagg ccacccggct gaagagaacc gccagaagaa gatacaccag acggaagaac
     2821 cggatctgct atctgcaaga gatcttcagc aacgagatgg ccaaggtgga cgacagcttc
     2881 ttccacagac tggaagagtc cttcctggtg gaagaggata agaagcacga gcggcacccc
     2941 atcttcggca acatcgtgga cgaggtggcc taccacgaga agtaccccac catctaccac
     3001 ctgagaaaga aactggtgga cagcaccgac aaggccgacc tgcggctgat ctatctggcc
     3061 ctggcccaca tgatcaagtt ccggggccac ttcctgatcg agggcgacct gaaccccgac
     3121 aacagcgacg tggacaagct gttcatccag ctggtgcaga cctacaacca gctgttcgag
     3181 gaaaacccca tcaacgccag cggcgtggac gccaaggcca tcctgtctgc cagactgagc
     3241 aagagcagac ggctggaaaa tctgatcgcc cagctgcccg gcgagaagaa gaatggcctg
     3301 ttcggaaacc tgattgccct gagcctgggc ctgaccccca acttcaagag caacttcgac
     3361 ctggccgagg atgccaaact gcagctgagc aaggacacct acgacgacga cctggacaac
     3421 ctgctggccc agatcggcga ccagtacgcc gacctgtttc tggccgccaa gaacctgtcc
     3481 gacgccatcc tgctgagcga catcctgaga gtgaacaccg agatcaccaa ggcccccctg
     3541 agcgcctcta tgatcaagag atacgacgag caccaccagg acctgaccct gctgaaagct
     3601 ctcgtgcggc agcagctgcc tgagaagtac aaagagattt tcttcgacca gagcaagaac
     3661 ggctacgccg gctacattga cggcggagcc agccaggaag agttctacaa gttcatcaag
     3721 cccatcctgg aaaagatgga cggcaccgag gaactgctcg tgaagctgaa cagagaggac
     3781 ctgctgcgga agcagcggac cttcgacaac ggcagcatcc cccaccagat ccacctggga
     3841 gagctgcacg ccattctgcg gcggcaggaa gatttttacc cattcctgaa ggacaaccgg
     3901 gaaaagatcg agaagatcct gaccttccgc atcccctact acgtgggccc tctggccagg
     3961 ggaaacagca gattcgcctg gatgaccaga aagagcgagg aaaccatcac cccctggaac
     4021 ttcgaggaag tggtggacaa gggcgcttcc gcccagagct tcatcgagcg gatgaccaac
     4081 ttcgataaga acctgcccaa cgagaaggtg ctgcccaagc acagcctgct gtacgagtac
     4141 ttcaccgtgt ataacgagct gaccaaagtg aaatacgtga ccgagggaat gagaaagccc
     4201 gccttcctga gcggcgagca gaaaaaggcc atcgtggacc tgctgttcaa gaccaaccgg
     4261 aaagtgaccg tgaagcagct gaaagaggac tacttcaaga aaatcgagtg cttcgactcc
     4321 gtggaaatct ccggcgtgga agatcggttc aacgcctccc tgggcacata ccacgatctg
     4381 ctgaaaatta tcaaggacaa ggacttcctg gacaatgagg aaaacgagga cattctggaa
     4441 gatatcgtgc tgaccctgac actgtttgag gacagagaga tgatcgagga acggctgaaa
     4501 acctatgccc acctgttcga cgacaaagtg atgaagcagc tgaagcggcg gagatacacc
     4561 ggctggggca ggctgagccg gaagctgatc aacggcatcc gggacaagca gtccggcaag
     4621 acaatcctgg atttcctgaa gtccgacggc ttcgccaaca gaaacttcat gcagctgatc
     4681 cacgacgaca gcctgacctt taaagaggac atccagaaag cccaggtgtc cggccagggc
     4741 gatagcctgc acgagcacat tgccaatctg gccggcagcc ccgccattaa gaagggcatc
     4801 ctgcagacag tgaaggtggt ggacgagctc gtgaaagtga tgggccggca caagcccgag
     4861 aacatcgtga tcgaaatggc cagagagaac cagaccaccc agaagggaca gaagaacagc
     4921 cgcgagagaa tgaagcggat cgaagagggc atcaaagagc tgggcagcca gatcctgaaa
     4981 gaacaccccg tggaaaacac ccagctgcag aacgagaagc tgtacctgta ctacctgcag
     5041 aatgggcggg atatgtacgt ggaccaggaa ctggacatca accggctgtc cgactacgat
     5101 gtggaccata tcgtgcctca gagctttctg aaggacgact ccatcgacaa caaggtgctg
     5161 accagaagcg acaagaaccg gggcaagagc gacaacgtgc cctccgaaga ggtcgtgaag
     5221 aagatgaaga actactggcg gcagctgctg aacgccaagc tgattaccca gagaaagttc
     5281 gacaatctga ccaaggccga gagaggcggc ctgagcgaac tggataaggc cggcttcatc
     5341 aagagacagc tggtggaaac ccggcagatc acaaagcacg tggcacagat cctggactcc
     5401 cggatgaaca ctaagtacga cgagaatgac aagctgatcc gggaagtgaa agtgatcacc
     5461 ctgaagtcca agctggtgtc cgatttccgg aaggatttcc agttttacaa agtgcgcgag
     5521 atcaacaact accaccacgc ccacgacgcc tacctgaacg ccgtcgtggg aaccgccctg
     5581 atcaaaaagt accctaagct ggaaagcgag ttcgtgtacg gcgactacaa ggtgtacgac
     5641 gtgcggaaga tgatcgccaa gagcgagcag gaaatcggca aggctaccgc caagtacttc
     5701 ttctacagca acatcatgaa ctttttcaag accgagatta ccctggccaa cggcgagatc
     5761 cggaagcggc ctctgatcga gacaaacggc gaaaccgggg agatcgtgtg ggataagggc
     5821 cgggattttg ccaccgtgcg gaaagtgctg agcatgcccc aagtgaatat cgtgaaaaag
     5881 accgaggtgc agacaggcgg cttcagcaaa gagtctatcc tgcccaagag gaacagcgat
     5941 aagctgatcg ccagaaagaa ggactgggac cctaagaagt acggcggctt cgacagcccc
     6001 accgtggcct attctgtgct ggtggtggcc aaagtggaaa agggcaagtc caagaaactg
     6061 aagagtgtga aagagctgct ggggatcacc atcatggaaa gaagcagctt cgagaagaat
     6121 cccatcgact ttctggaagc caagggctac aaagaagtga aaaaggacct gatcatcaag
     6181 ctgcctaagt actccctgtt cgagctggaa aacggccgga agagaatgct ggcctctgcc
     6241 ggcgaactgc agaagggaaa cgaactggcc ctgccctcca aatatgtgaa cttcctgtac
     6301 ctggccagcc actatgagaa gctgaagggc tcccccgagg ataatgagca gaaacagctg
     6361 tttgtggaac agcacaagca ctacctggac gagatcatcg agcagatcag cgagttctcc
     6421 aagagagtga tcctggccga cgctaatctg gacaaagtgc tgtccgccta caacaagcac
     6481 cgggataagc ccatcagaga gcaggccgag aatatcatcc acctgtttac cctgaccaat
     6541 ctgggagccc ctgccgcctt caagtacttt gacaccacca tcgaccggaa gaggtacacc
     6601 agcaccaaag aggtgctgga cgccaccctg atccaccaga gcatcaccgg cctgtacgag
     6661 acacggatcg acctgtctca gctgggaggc gacaagcgac ctgccgccac aaagaaggct
     6721 ggacaggcta agaagaagaa agattacaaa gacgatgacg ataagggatc cggcgcaaca
     6781 aacttctctc tgctgaaaca agccggagat gtcgaagaga atcctggacc gaccgagtac
     6841 aagcccacgg tgcgcctcgc cacccgcgac gacgtcccca gggccgtacg caccctcgcc
     6901 gccgcgttcg ccgactaccc cgccacgcgc cacaccgtcg atccggaccg ccacatcgag
     6961 cgggtcaccg agctgcaaga actcttcctc acgcgcgtcg ggctcgacat cggcaaggtg
     7021 tgggtcgcgg acgacggcgc cgcggtggcg gtctggacca cgccggagag cgtcgaagcg
     7081 ggggcggtgt tcgccgagat cggcccgcgc atggccgagt tgagcggttc ccggctggcc
     7141 gcgcagcaac agatggaagg cctcctggcg ccgcaccggc ccaaggagcc cgcgtggttc
     7201 ctggccaccg tcggagtctc gcccgaccac cagggcaagg gtctgggcag cgccgtcgtg
     7261 ctccccggag tggaggcggc cgagcgcgcc ggggtgcccg ccttcctgga gacctccgcg
     7321 ccccgcaacc tccccttcta cgagcggctc ggcttcaccg tcaccgccga cgtcgaggtg
     7381 cccgaaggac cgcgcacctg gtgcatgacc cgcaagcccg gtgcctgaac gcgttaagtc
     7441 gacaatcaac ctctggatta caaaatttgt gaaagattga ctggtattct taactatgtt
     7501 gctcctttta cgctatgtgg atacgctgct ttaatgcctt tgtatcatgc tattgcttcc
     7561 cgtatggctt tcattttctc ctccttgtat aaatcctggt tgctgtctct ttatgaggag
     7621 ttgtggcccg ttgtcaggca acgtggcgtg gtgtgcactg tgtttgctga cgcaaccccc
     7681 actggttggg gcattgccac cacctgtcag ctcctttccg ggactttcgc tttccccctc
     7741 cctattgcca cggcggaact catcgccgcc tgccttgccc gctgctggac aggggctcgg
     7801 ctgttgggca ctgacaattc cgtggtgttg tcggggaaat catcgtcctt tccttggctg
     7861 ctcgcctgtg ttgccacctg gattctgcgc gggacgtcct tctgctacgt cccttcggcc
     7921 ctcaatccag cggaccttcc ttcccgcggc ctgctgccgg ctctgcggcc tcttccgcgt
     7981 cttcgccttc gccctcagac gagtcggatc tccctttggg ccgcctcccc gcgtcgactt
     8041 taagaccaat gacttacaag gcagctgtag atcttagcca ctttttaaaa gaaaaggggg
     8101 gactggaagg gctaattcac tcccaacgaa gacaagatct gctttttgct tgtactgggt
     8161 ctctctggtt agaccagatc tgagcctggg agctctctgg ctaactaggg aacccactgc
     8221 ttaagcctca ataaagcttg ccttgagtgc ttcaagtagt gtgtgcccgt ctgttgtgtg
     8281 actctggtaa ctagagatcc ctcagaccct tttagtcagt gtggaaaatc tctagcaggg
     8341 cccgtttaaa cccgctgatc agcctcgact gtgccttcta gttgccagcc atctgttgtt
     8401 tgcccctccc ccgtgccttc cttgaccctg gaaggtgcca ctcccactgt cctttcctaa
     8461 taaaatgagg aaattgcatc gcattgtctg agtaggtgtc attctattct ggggggtggg
     8521 gtggggcagg acagcaaggg ggaggattgg gaagacaata gcaggcatgc tggggatgcg
     8581 gtgggctcta tggcttctga ggcggaaaga accagctggg gctctagggg gtatccccac
     8641 gcgccctgta gcggcgcatt aagcgcggcg ggtgtggtgg ttacgcgcag cgtgaccgct
     8701 acacttgcca gcgccctagc gcccgctcct ttcgctttct tcccttcctt tctcgccacg
     8761 ttcgccggct ttccccgtca agctctaaat cgggggctcc ctttagggtt ccgatttagt
     8821 gctttacggc acctcgaccc caaaaaactt gattagggtg atggttcacg tagtgggcca
     8881 tcgccctgat agacggtttt tcgccctttg acgttggagt ccacgttctt taatagtgga
     8941 ctcttgttcc aaactggaac aacactcaac cctatctcgg tctattcttt tgatttataa
     9001 gggattttgc cgatttcggc ctattggtta aaaaatgagc tgatttaaca aaaatttaac
     9061 gcgaattaat tctgtggaat gtgtgtcagt tagggtgtgg aaagtcccca ggctccccag
     9121 caggcagaag tatgcaaagc atgcatctca attagtcagc aaccaggtgt ggaaagtccc
     9181 caggctcccc agcaggcaga agtatgcaaa gcatgcatct caattagtca gcaaccatag
     9241 tcccgcccct aactccgccc atcccgcccc taactccgcc cagttccgcc cattctccgc
     9301 cccatggctg actaattttt tttatttatg cagaggccga ggccgcctct gcctctgagc
     9361 tattccagaa gtagtgagga ggcttttttg gaggcctagg cttttgcaaa aagctcccgg
     9421 gagcttgtat atccattttc ggatctgatc agcacgtgtt gacaattaat catcggcata
     9481 gtatatcggc atagtataat acgacaaggt gaggaactaa accatggcca agttgaccag
     9541 tgccgttccg gtgctcaccg cgcgcgacgt cgccggagcg gtcgagttct ggaccgaccg
     9601 gctcgggttc tcccgggact tcgtggagga cgacttcgcc ggtgtggtcc gggacgacgt
     9661 gaccctgttc atcagcgcgg tccaggacca ggtggtgccg gacaacaccc tggcctgggt
     9721 gtgggtgcgc ggcctggacg agctgtacgc cgagtggtcg gaggtcgtgt ccacgaactt
     9781 ccgggacgcc tccgggccgg ccatgaccga gatcggcgag cagccgtggg ggcgggagtt
     9841 cgccctgcgc gacccggccg gcaactgcgt gcacttcgtg gccgaggagc aggactgaca
     9901 cgtgctacga gatttcgatt ccaccgccgc cttctatgaa aggttgggct tcggaatcgt
     9961 tttccgggac gccggctgga tgatcctcca gcgcggggat ctcatgctgg agttcttcgc
    10021 ccaccccaac ttgtttattg cagcttataa tggttacaaa taaagcaata gcatcacaaa
    10081 tttcacaaat aaagcatttt tttcactgca ttctagttgt ggtttgtcca aactcatcaa
    10141 tgtatcttat catgtctgta taccgtcgac ctctagctag agcttggcgt aatcatggtc
    10201 atagctgttt cctgtgtgaa attgttatcc gctcacaatt ccacacaaca tacgagccgg
    10261 aagcataaag tgtaaagcct ggggtgccta atgagtgagc taactcacat taattgcgtt
    10321 gcgctcactg cccgctttcc agtcgggaaa cctgtcgtgc cagctgcatt aatgaatcgg
    10381 ccaacgcgcg gggagaggcg gtttgcgtat tgggcgctct tccgcttcct cgctcactga
    10441 ctcgctgcgc tcggtcgttc ggctgcggcg agcggtatca gctcactcaa aggcggtaat
    10501 acggttatcc acagaatcag gggataacgc aggaaagaac atgtgagcaa aaggccagca
    10561 aaaggccagg aaccgtaaaa aggccgcgtt gctggcgttt ttccataggc tccgcccccc
    10621 tgacgagcat cacaaaaatc gacgctcaag tcagaggtgg cgaaacccga caggactata
    10681 aagataccag gcgtttcccc ctggaagctc cctcgtgcgc tctcctgttc cgaccctgcc
    10741 gcttaccgga tacctgtccg cctttctccc ttcgggaagc gtggcgcttt ctcatagctc
    10801 acgctgtagg tatctcagtt cggtgtaggt cgttcgctcc aagctgggct gtgtgcacga
    10861 accccccgtt cagcccgacc gctgcgcctt atccggtaac tatcgtcttg agtccaaccc
    10921 ggtaagacac gacttatcgc cactggcagc agccactggt aacaggatta gcagagcgag
    10981 gtatgtaggc ggtgctacag agttcttgaa gtggtggcct aactacggct acactagaag
    11041 aacagtattt ggtatctgcg ctctgctgaa gccagttacc ttcggaaaaa gagttggtag
    11101 ctcttgatcc ggcaaacaaa ccaccgctgg tagcggtggt ttttttgttt gcaagcagca
    11161 gattacgcgc agaaaaaaag gatctcaaga agatcctttg atcttttcta cggggtctga
    11221 cgctcagtgg aacgaaaact cacgttaagg gattttggtc atgagattat caaaaaggat
    11281 cttcacctag atccttttaa attaaaaatg aagttttaaa tcaatctaaa gtatatatga
    11341 gtaaacttgg tctgacagtt accaatgctt aatcagtgag gcacctatct cagcgatctg
    11401 tctatttcgt tcatccatag ttgcctgact ccccgtcgtg tagataacta cgatacggga
    11461 gggcttacca tctggcccca gtgctgcaat gataccgcga gacccacgct caccggctcc
    11521 agatttatca gcaataaacc agccagccgg aagggccgag cgcagaagtg gtcctgcaac
    11581 tttatccgcc tccatccagt ctattaattg ttgccgggaa gctagagtaa gtagttcgcc
    11641 agttaatagt ttgcgcaacg ttgttgccat tgctacaggc atcgtggtgt cacgctcgtc
    11701 gtttggtatg gcttcattca gctccggttc ccaacgatca aggcgagtta catgatcccc
    11761 catgttgtgc aaaaaagcgg ttagctcctt cggtcctccg atcgttgtca gaagtaagtt
    11821 ggccgcagtg ttatcactca tggttatggc agcactgcat aattctctta ctgtcatgcc
    11881 atccgtaaga tgcttttctg tgactggtga gtactcaacc aagtcattct gagaatagtg
    11941 tatgcggcga ccgagttgct cttgcccggc gtcaatacgg gataataccg cgccacatag
    12001 cagaacttta aaagtgctca tcattggaaa acgttcttcg gggcgaaaac tctcaaggat
    12061 cttaccgctg ttgagatcca gttcgatgta acccactcgt gcacccaact gatcttcagc
    12121 atcttttact ttcaccagcg tttctgggtg agcaaaaaca ggaaggcaaa atgccgcaaa
    12181 aaagggaata agggcgacac ggaaatgttg aatactcata ctcttccttt ttcaatatta
    12241 ttgaagcatt tatcagggtt attgtctcat gagcggatac atatttgaat gtatttagaa
    12301 aaataaacaa ataggggttc cgcgcacatt tccccgaaaa gtgccacctg acgtcgacgg
    12361 atcgggagat ctcccgatcc cctatggtgc actctcagta caatctgctc tgatgccgca
    12421 tagttaagcc agtatctgct ccctgcttgt gtgttggagg tcgctgagta gtgcgcgagc
    12481 aaaatttaag ctacaacaag gcaaggcttg accgacaatt gcatgaagaa tctgcttagg
    12541 gttaggcgtt ttgcgctgct tcgcgatgta cgggccagat atacgcgttg acattgatta
    12601 ttgactagtt attaatagta atcaattacg gggtcattag ttcatagccc atatatggag
    12661 ttccgcgtta cataacttac ggtaaatggc ccgcctggct gaccgcccaa cgacccccgc
    12721 ccattgacgt caataatgac gtatgttccc atagtaacgc caatagggac tttccattga
    12781 cgtcaatggg tggagtattt acggtaaact gcccacttgg cagtacatca agtgtatcat
    12841 atgccaagta cgccccctat tgacgtcaat gacggtaaat ggcccgcctg gcattatgcc
    12901 cagtacatga ccttatggga ctttcctact tggcagtaca tctacgtatt agtcatcgct
    12961 attaccatgg tgatgcggtt ttggcagtac atcaatgggc gtggatagcg gtt
//
