LOCUS       sequence-413631-       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-2
FEATURES             Location/Qualifiers
     source          1..13013
                     /mol_type="other DNA"
                     /organism="synthetic DNA construct"
     promoter        6..204
                     /label=CMV promoter
                     /note="human cytomegalovirus (CMV) immediate early 
                     promoter"
     primer_bind     159..179
                     /label=CMV-F
                     /note="Human CMV immediate early promoter, forward primer"
     LTR             222..402
                     /label=5' LTR (truncated)
                     /note="truncated 5' long terminal repeat (LTR) from HIV-1"
     misc_feature    449..574
                     /label=HIV-1 Psi
                     /note="packaging signal of human immunodeficiency virus 
                     type 1"
     misc_feature    1067..1300
                     /label=RRE
                     /note="The Rev response element (RRE) of HIV-1 allows for 
                     Rev-dependent mRNA export from the nucleus to the 
                     cytoplasm."
     CDS             1485..1529
                     /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    1827..1944
                     /label=cPPT/CTS
                     /note="central polypurine tract and central termination 
                     sequence of HIV-1"
     promoter        1995..2243
                     /label=U6 promoter
                     /note="RNA polymerase III promoter for human U6 snRNA 
                     (Domitrovich & Kunkel, 2003)"
     primer_bind     1995..2015
                     /label=hU6-F
                     /note="Human U6 promoter, forward primer"
     primer_bind     2166..2185
                     /label=LKO.1 5'
                     /note="Human U6 promoter, forward primer"
     misc_RNA        2265..2340
                     /label=gRNA scaffold
                     /note="guide RNA scaffold for the Streptococcus pyogenes 
                     CRISPR/Cas9 system"
     promoter        2402..2613
                     /label=EF-1-alpha core promoter
                     /note="core promoter for human elongation factor 
                     EF-1-alpha"
     regulatory      2632..2641
                     /label=Kozak sequence
                     /note="vertebrate consensus sequence for strong initiation 
                     of translation (Kozak, 1987)"
                     /regulatory_class="other"
     CDS             2638..6741
                     /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             2638..6741
                     /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             6742..6789
                     /codon_start=1
                     /product="bipartite nuclear localization signal from 
                     nucleoplasmin"
                     /label=nucleoplasmin NLS
                     /translation="KRPAATKKAGQAKKKK"
     CDS             6790..6813
                     /codon_start=1
                     /product="FLAG(R) epitope tag, followed by an enterokinase 
                     cleavage site"
                     /label=FLAG
                     /translation="DYKDDDDK"
     CDS             6823..6879
                     /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             6880..7476
                     /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     7373..7393
                     /label=Puro-F
                     /note="Puromycin resistance gene, forward primer"
     misc_feature    7492..8080
                     /label=WPRE
                     /note="woodchuck hepatitis virus posttranscriptional 
                     regulatory element"
     primer_bind     complement(7545..7565)
                     /label=WPRE-R
                     /note="WPRE, reverse primer"
     CDS             complement(7963..7974)
                     /codon_start=1
                     /product="Factor Xa recognition and cleavage site"
                     /label=Factor Xa site
                     /translation="IEGR"
     LTR             8152..8385
                     /label=3' LTR (Delta-U3)
                     /note="self-inactivating 3' long terminal repeat (LTR) from
                     HIV-1"
     primer_bind     complement(8411..8428)
                     /label=BGH-rev
                     /note="Bovine growth hormone terminator, reverse primer. 
                     Also called BGH reverse"
     polyA_signal    8417..8641
                     /label=bGH poly(A) signal
                     /note="bovine growth hormone polyadenylation signal"
     rep_origin      8687..9115
                     /direction=RIGHT
                     /label=f1 ori
                     /note="f1 bacteriophage origin of replication; arrow 
                     indicates direction of (+) strand synthesis"
     primer_bind     complement(8774..8793)
                     /label=F1ori-R
                     /note="F1 origin, reverse primer"
     primer_bind     8984..9005
                     /label=F1ori-F
                     /note="F1 origin, forward primer"
     primer_bind     complement(9124..9144)
                     /label=pBABE 3'
                     /note="SV40 enhancer, reverse primer for pBABE vectors"
     promoter        9129..9458
                     /label=SV40 promoter
                     /note="SV40 enhancer and early promoter"
     rep_origin      9309..9444
                     /label=SV40 ori
                     /note="SV40 origin of replication"
     primer_bind     9371..9390
                     /label=SV40pro-F
                     /note="SV40 promoter/origin, forward primer"
     promoter        9506..9553
                     /label=EM7 promoter
                     /note="synthetic bacterial promoter "
     CDS             9572..9946
                     /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    10076..10197
                     /label=SV40 poly(A) signal
                     /note="SV40 polyadenylation signal"
     primer_bind     complement(10113..10132)
                     /label=SV40pA-R
                     /note="SV40 polyA, reverse primer"
     primer_bind     10167..10186
                     /label=EBV-rev
                     /note="SV40 polyA terminator, reverse primer"
     primer_bind     complement(10246..10262)
                     /label=M13 rev
                     /note="common sequencing primer, one of multiple similar 
                     variants"
     primer_bind     complement(10246..10262)
                     /label=M13 Reverse
                     /note="In lacZ gene. Also called M13-rev"
     primer_bind     complement(10259..10281)
                     /label=M13/pUC Reverse
                     /note="In lacZ gene"
     protein_bind    10270..10286
                     /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(10294..10300,10301..10318,10319..10324))
                     /label=lac promoter
                     /note="promoter for the E. coli lac operon"
     protein_bind    10339..10360
                     /label=CAP binding site
                     /bound_moiety="E. coli catabolite activator protein"
                     /note="CAP binding activates transcription in the presence 
                     of cAMP."
     primer_bind     complement(10477..10494)
                     /label=L4440
                     /note="L4440 vector, forward primer"
     rep_origin      complement(10648..11236)
                     /direction=LEFT
                     /label=ori
                     /note="high-copy-number ColE1/pMB1/pBR322/pUC origin of 
                     replication"
     primer_bind     complement(10728..10747)
                     /label=pBR322ori-F
                     /note="pBR322 origin, forward primer"
     CDS             complement(join(11407..12198,12199..12267))
                     /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     12030..12049
                     /label=Amp-R
                     /note="Ampicillin resistance gene, reverse primer"
     promoter        complement(12268..12372)
                     /gene="bla"
                     /label=AmpR promoter
     primer_bind     complement(12447..12466)
                     /label=pRS-marker
                     /note="pRS vectors, use to sequence yeast selectable 
                     marker"
     enhancer        join(12638..13013,1..4)
                     /label=CMV enhancer
                     /note="human cytomegalovirus immediate early enhancer"
ORIGIN
        1 catggtgatg cggttttggc agtacatcaa tgggcgtgga tagcggtttg actcacgggg
       61 atttccaagt ctccacccca ttgacgtcaa tgggagtttg ttttggcacc aaaatcaacg
      121 ggactttcca aaatgtcgta acaactccgc cccattgacg caaatgggcg gtaggcgtgt
      181 acggtgggag gtctatataa gcagcgcgtt ttgcctgtac tgggtctctc tggttagacc
      241 agatctgagc ctgggagctc tctggctaac tagggaaccc actgcttaag cctcaataaa
      301 gcttgccttg agtgcttcaa gtagtgtgtg cccgtctgtt gtgtgactct ggtaactaga
      361 gatccctcag acccttttag tcagtgtgga aaatctctag cagtggcgcc cgaacaggga
      421 cttgaaagcg aaagggaaac cagaggagct ctctcgacgc aggactcggc ttgctgaagc
      481 gcgcacggca agaggcgagg ggcggcgact ggtgagtacg ccaaaaattt tgactagcgg
      541 aggctagaag gagagagatg ggtgcgagag cgtcagtatt aagcggggga gaattagatc
      601 gcgatgggaa aaaattcggt taaggccagg gggaaagaaa aaatataaat taaaacatat
      661 agtatgggca agcagggagc tagaacgatt cgcagttaat cctggcctgt tagaaacatc
      721 agaaggctgt agacaaatac tgggacagct acaaccatcc cttcagacag gatcagaaga
      781 acttagatca ttatataata cagtagcaac cctctattgt gtgcatcaaa ggatagagat
      841 aaaagacacc aaggaagctt tagacaagat agaggaagag caaaacaaaa gtaagaccac
      901 cgcacagcaa gcggccgctg atcttcagac ctggaggagg agatatgagg gacaattgga
      961 gaagtgaatt atataaatat aaagtagtaa aaattgaacc attaggagta gcacccacca
     1021 aggcaaagag aagagtggtg cagagagaaa aaagagcagt gggaatagga gctttgttcc
     1081 ttgggttctt gggagcagca ggaagcacta tgggcgcagc gtcaatgacg ctgacggtac
     1141 aggccagaca attattgtct ggtatagtgc agcagcagaa caatttgctg agggctattg
     1201 aggcgcaaca gcatctgttg caactcacag tctggggcat caagcagctc caggcaagaa
     1261 tcctggctgt ggaaagatac ctaaaggatc aacagctcct ggggatttgg ggttgctctg
     1321 gaaaactcat ttgcaccact gctgtgcctt ggaatgctag ttggagtaat aaatctctgg
     1381 aacagatttg gaatcacacg acctggatgg agtgggacag agaaattaac aattacacaa
     1441 gcttaataca ctccttaatt gaagaatcgc aaaaccagca agaaaagaat gaacaagaat
     1501 tattggaatt agataaatgg gcaagtttgt ggaattggtt taacataaca aattggctgt
     1561 ggtatataaa attattcata atgatagtag gaggcttggt aggtttaaga atagtttttg
     1621 ctgtactttc tatagtgaat agagttaggc agggatattc accattatcg tttcagaccc
     1681 acctcccaac cccgagggga cccgacaggc ccgaaggaat agaagaagaa ggtggagaga
     1741 gagacagaga cagatccatt cgattagtga acggatcggc actgcgtgcg ccaattctgc
     1801 agacaaatgg cagtattcat ccacaatttt aaaagaaaag gggggattgg ggggtacagt
     1861 gcaggggaaa gaatagtaga cataatagca acagacatac aaactaaaga attacaaaaa
     1921 caaattacaa aaattcaaaa ttttcgggtt tattacaggg acagcagaga tccagtttgg
     1981 ttaattaagg taccgagggc ctatttccca tgattccttc atatttgcat atacgataca
     2041 aggctgttag agagataatt agaattaatt tgactgtaaa cacaaagata ttagtacaaa
     2101 atacgtgacg tagaaagtaa taatttcttg ggtagtttgc agttttaaaa ttatgtttta
     2161 aaatggacta tcatatgctt accgtaactt gaaagtattt cgatttcttg gctttatata
     2221 tcttgtggaa aggacgaaac accgggaact ctgagtcata gcgtgtttta gagctagaaa
     2281 tagcaagtta aaataaggct agtccgttat caacttgaaa aagtggcacc gagtcggtgc
     2341 ttttttgaat tcgctagcta ggtcttgaaa ggagtgggaa ttggctccgg tgcccgtcag
     2401 tgggcagagc gcacatcgcc cacagtcccc gagaagttgg ggggaggggt cggcaattga
     2461 tccggtgcct agagaaggtg gcgcggggta aactgggaaa gtgatgtcgt gtactggctc
     2521 cgcctttttc ccgagggtgg gggagaaccg tatataagtg cagtagtcgc cgtgaacgtt
     2581 ctttttcgca acgggtttgc cgccagaaca caggaccggt tctagagcgc tgccaccatg
     2641 gacaagaagt acagcatcgg cctggacatc ggcaccaact ctgtgggctg ggccgtgatc
     2701 accgacgagt acaaggtgcc cagcaagaaa ttcaaggtgc tgggcaacac cgaccggcac
     2761 agcatcaaga agaacctgat cggagccctg ctgttcgaca gcggcgaaac agccgaggcc
     2821 acccggctga agagaaccgc cagaagaaga tacaccagac ggaagaaccg gatctgctat
     2881 ctgcaagaga tcttcagcaa cgagatggcc aaggtggacg acagcttctt ccacagactg
     2941 gaagagtcct tcctggtgga agaggataag aagcacgagc ggcaccccat cttcggcaac
     3001 atcgtggacg aggtggccta ccacgagaag taccccacca tctaccacct gagaaagaaa
     3061 ctggtggaca gcaccgacaa ggccgacctg cggctgatct atctggccct ggcccacatg
     3121 atcaagttcc ggggccactt cctgatcgag ggcgacctga accccgacaa cagcgacgtg
     3181 gacaagctgt tcatccagct ggtgcagacc tacaaccagc tgttcgagga aaaccccatc
     3241 aacgccagcg gcgtggacgc caaggccatc ctgtctgcca gactgagcaa gagcagacgg
     3301 ctggaaaatc tgatcgccca gctgcccggc gagaagaaga atggcctgtt cggaaacctg
     3361 attgccctga gcctgggcct gacccccaac ttcaagagca acttcgacct ggccgaggat
     3421 gccaaactgc agctgagcaa ggacacctac gacgacgacc tggacaacct gctggcccag
     3481 atcggcgacc agtacgccga cctgtttctg gccgccaaga acctgtccga cgccatcctg
     3541 ctgagcgaca tcctgagagt gaacaccgag atcaccaagg cccccctgag cgcctctatg
     3601 atcaagagat acgacgagca ccaccaggac ctgaccctgc tgaaagctct cgtgcggcag
     3661 cagctgcctg agaagtacaa agagattttc ttcgaccaga gcaagaacgg ctacgccggc
     3721 tacattgacg gcggagccag ccaggaagag ttctacaagt tcatcaagcc catcctggaa
     3781 aagatggacg gcaccgagga actgctcgtg aagctgaaca gagaggacct gctgcggaag
     3841 cagcggacct tcgacaacgg cagcatcccc caccagatcc acctgggaga gctgcacgcc
     3901 attctgcggc ggcaggaaga tttttaccca ttcctgaagg acaaccggga aaagatcgag
     3961 aagatcctga ccttccgcat cccctactac gtgggccctc tggccagggg aaacagcaga
     4021 ttcgcctgga tgaccagaaa gagcgaggaa accatcaccc cctggaactt cgaggaagtg
     4081 gtggacaagg gcgcttccgc ccagagcttc atcgagcgga tgaccaactt cgataagaac
     4141 ctgcccaacg agaaggtgct gcccaagcac agcctgctgt acgagtactt caccgtgtat
     4201 aacgagctga ccaaagtgaa atacgtgacc gagggaatga gaaagcccgc cttcctgagc
     4261 ggcgagcaga aaaaggccat cgtggacctg ctgttcaaga ccaaccggaa agtgaccgtg
     4321 aagcagctga aagaggacta cttcaagaaa atcgagtgct tcgactccgt ggaaatctcc
     4381 ggcgtggaag atcggttcaa cgcctccctg ggcacatacc acgatctgct gaaaattatc
     4441 aaggacaagg acttcctgga caatgaggaa aacgaggaca ttctggaaga tatcgtgctg
     4501 accctgacac tgtttgagga cagagagatg atcgaggaac ggctgaaaac ctatgcccac
     4561 ctgttcgacg acaaagtgat gaagcagctg aagcggcgga gatacaccgg ctggggcagg
     4621 ctgagccgga agctgatcaa cggcatccgg gacaagcagt ccggcaagac aatcctggat
     4681 ttcctgaagt ccgacggctt cgccaacaga aacttcatgc agctgatcca cgacgacagc
     4741 ctgaccttta aagaggacat ccagaaagcc caggtgtccg gccagggcga tagcctgcac
     4801 gagcacattg ccaatctggc cggcagcccc gccattaaga agggcatcct gcagacagtg
     4861 aaggtggtgg acgagctcgt gaaagtgatg ggccggcaca agcccgagaa catcgtgatc
     4921 gaaatggcca gagagaacca gaccacccag aagggacaga agaacagccg cgagagaatg
     4981 aagcggatcg aagagggcat caaagagctg ggcagccaga tcctgaaaga acaccccgtg
     5041 gaaaacaccc agctgcagaa cgagaagctg tacctgtact acctgcagaa tgggcgggat
     5101 atgtacgtgg accaggaact ggacatcaac cggctgtccg actacgatgt ggaccatatc
     5161 gtgcctcaga gctttctgaa ggacgactcc atcgacaaca aggtgctgac cagaagcgac
     5221 aagaaccggg gcaagagcga caacgtgccc tccgaagagg tcgtgaagaa gatgaagaac
     5281 tactggcggc agctgctgaa cgccaagctg attacccaga gaaagttcga caatctgacc
     5341 aaggccgaga gaggcggcct gagcgaactg gataaggccg gcttcatcaa gagacagctg
     5401 gtggaaaccc ggcagatcac aaagcacgtg gcacagatcc tggactcccg gatgaacact
     5461 aagtacgacg agaatgacaa gctgatccgg gaagtgaaag tgatcaccct gaagtccaag
     5521 ctggtgtccg atttccggaa ggatttccag ttttacaaag tgcgcgagat caacaactac
     5581 caccacgccc acgacgccta cctgaacgcc gtcgtgggaa ccgccctgat caaaaagtac
     5641 cctaagctgg aaagcgagtt cgtgtacggc gactacaagg tgtacgacgt gcggaagatg
     5701 atcgccaaga gcgagcagga aatcggcaag gctaccgcca agtacttctt ctacagcaac
     5761 atcatgaact ttttcaagac cgagattacc ctggccaacg gcgagatccg gaagcggcct
     5821 ctgatcgaga caaacggcga aaccggggag atcgtgtggg ataagggccg ggattttgcc
     5881 accgtgcgga aagtgctgag catgccccaa gtgaatatcg tgaaaaagac cgaggtgcag
     5941 acaggcggct tcagcaaaga gtctatcctg cccaagagga acagcgataa gctgatcgcc
     6001 agaaagaagg actgggaccc taagaagtac ggcggcttcg acagccccac cgtggcctat
     6061 tctgtgctgg tggtggccaa agtggaaaag ggcaagtcca agaaactgaa gagtgtgaaa
     6121 gagctgctgg ggatcaccat catggaaaga agcagcttcg agaagaatcc catcgacttt
     6181 ctggaagcca agggctacaa agaagtgaaa aaggacctga tcatcaagct gcctaagtac
     6241 tccctgttcg agctggaaaa cggccggaag agaatgctgg cctctgccgg cgaactgcag
     6301 aagggaaacg aactggccct gccctccaaa tatgtgaact tcctgtacct ggccagccac
     6361 tatgagaagc tgaagggctc ccccgaggat aatgagcaga aacagctgtt tgtggaacag
     6421 cacaagcact acctggacga gatcatcgag cagatcagcg agttctccaa gagagtgatc
     6481 ctggccgacg ctaatctgga caaagtgctg tccgcctaca acaagcaccg ggataagccc
     6541 atcagagagc aggccgagaa tatcatccac ctgtttaccc tgaccaatct gggagcccct
     6601 gccgccttca agtactttga caccaccatc gaccggaaga ggtacaccag caccaaagag
     6661 gtgctggacg ccaccctgat ccaccagagc atcaccggcc tgtacgagac acggatcgac
     6721 ctgtctcagc tgggaggcga caagcgacct gccgccacaa agaaggctgg acaggctaag
     6781 aagaagaaag attacaaaga cgatgacgat aagggatccg gcgcaacaaa cttctctctg
     6841 ctgaaacaag ccggagatgt cgaagagaat cctggaccga ccgagtacaa gcccacggtg
     6901 cgcctcgcca cccgcgacga cgtccccagg gccgtacgca ccctcgccgc cgcgttcgcc
     6961 gactaccccg ccacgcgcca caccgtcgat ccggaccgcc acatcgagcg ggtcaccgag
     7021 ctgcaagaac tcttcctcac gcgcgtcggg ctcgacatcg gcaaggtgtg ggtcgcggac
     7081 gacggcgccg cggtggcggt ctggaccacg ccggagagcg tcgaagcggg ggcggtgttc
     7141 gccgagatcg gcccgcgcat ggccgagttg agcggttccc ggctggccgc gcagcaacag
     7201 atggaaggcc tcctggcgcc gcaccggccc aaggagcccg cgtggttcct ggccaccgtc
     7261 ggagtctcgc ccgaccacca gggcaagggt ctgggcagcg ccgtcgtgct ccccggagtg
     7321 gaggcggccg agcgcgccgg ggtgcccgcc ttcctggaga cctccgcgcc ccgcaacctc
     7381 cccttctacg agcggctcgg cttcaccgtc accgccgacg tcgaggtgcc cgaaggaccg
     7441 cgcacctggt gcatgacccg caagcccggt gcctgaacgc gttaagtcga caatcaacct
     7501 ctggattaca aaatttgtga aagattgact ggtattctta actatgttgc tccttttacg
     7561 ctatgtggat acgctgcttt aatgcctttg tatcatgcta ttgcttcccg tatggctttc
     7621 attttctcct ccttgtataa atcctggttg ctgtctcttt atgaggagtt gtggcccgtt
     7681 gtcaggcaac gtggcgtggt gtgcactgtg tttgctgacg caacccccac tggttggggc
     7741 attgccacca cctgtcagct cctttccggg actttcgctt tccccctccc tattgccacg
     7801 gcggaactca tcgccgcctg ccttgcccgc tgctggacag gggctcggct gttgggcact
     7861 gacaattccg tggtgttgtc ggggaaatca tcgtcctttc cttggctgct cgcctgtgtt
     7921 gccacctgga ttctgcgcgg gacgtccttc tgctacgtcc cttcggccct caatccagcg
     7981 gaccttcctt cccgcggcct gctgccggct ctgcggcctc ttccgcgtct tcgccttcgc
     8041 cctcagacga gtcggatctc cctttgggcc gcctccccgc gtcgacttta agaccaatga
     8101 cttacaaggc agctgtagat cttagccact ttttaaaaga aaagggggga ctggaagggc
     8161 taattcactc ccaacgaaga caagatctgc tttttgcttg tactgggtct ctctggttag
     8221 accagatctg agcctgggag ctctctggct aactagggaa cccactgctt aagcctcaat
     8281 aaagcttgcc ttgagtgctt caagtagtgt gtgcccgtct gttgtgtgac tctggtaact
     8341 agagatccct cagacccttt tagtcagtgt ggaaaatctc tagcagggcc cgtttaaacc
     8401 cgctgatcag cctcgactgt gccttctagt tgccagccat ctgttgtttg cccctccccc
     8461 gtgccttcct tgaccctgga aggtgccact cccactgtcc tttcctaata aaatgaggaa
     8521 attgcatcgc attgtctgag taggtgtcat tctattctgg ggggtggggt ggggcaggac
     8581 agcaaggggg aggattggga agacaatagc aggcatgctg gggatgcggt gggctctatg
     8641 gcttctgagg cggaaagaac cagctggggc tctagggggt atccccacgc gccctgtagc
     8701 ggcgcattaa gcgcggcggg tgtggtggtt acgcgcagcg tgaccgctac acttgccagc
     8761 gccctagcgc ccgctccttt cgctttcttc ccttcctttc tcgccacgtt cgccggcttt
     8821 ccccgtcaag ctctaaatcg ggggctccct ttagggttcc gatttagtgc tttacggcac
     8881 ctcgacccca aaaaacttga ttagggtgat ggttcacgta gtgggccatc gccctgatag
     8941 acggtttttc gccctttgac gttggagtcc acgttcttta atagtggact cttgttccaa
     9001 actggaacaa cactcaaccc tatctcggtc tattcttttg atttataagg gattttgccg
     9061 atttcggcct attggttaaa aaatgagctg atttaacaaa aatttaacgc gaattaattc
     9121 tgtggaatgt gtgtcagtta gggtgtggaa agtccccagg ctccccagca ggcagaagta
     9181 tgcaaagcat gcatctcaat tagtcagcaa ccaggtgtgg aaagtcccca ggctccccag
     9241 caggcagaag tatgcaaagc atgcatctca attagtcagc aaccatagtc ccgcccctaa
     9301 ctccgcccat cccgccccta actccgccca gttccgccca ttctccgccc catggctgac
     9361 taattttttt tatttatgca gaggccgagg ccgcctctgc ctctgagcta ttccagaagt
     9421 agtgaggagg cttttttgga ggcctaggct tttgcaaaaa gctcccggga gcttgtatat
     9481 ccattttcgg atctgatcag cacgtgttga caattaatca tcggcatagt atatcggcat
     9541 agtataatac gacaaggtga ggaactaaac catggccaag ttgaccagtg ccgttccggt
     9601 gctcaccgcg cgcgacgtcg ccggagcggt cgagttctgg accgaccggc tcgggttctc
     9661 ccgggacttc gtggaggacg acttcgccgg tgtggtccgg gacgacgtga ccctgttcat
     9721 cagcgcggtc caggaccagg tggtgccgga caacaccctg gcctgggtgt gggtgcgcgg
     9781 cctggacgag ctgtacgccg agtggtcgga ggtcgtgtcc acgaacttcc gggacgcctc
     9841 cgggccggcc atgaccgaga tcggcgagca gccgtggggg cgggagttcg ccctgcgcga
     9901 cccggccggc aactgcgtgc acttcgtggc cgaggagcag gactgacacg tgctacgaga
     9961 tttcgattcc accgccgcct tctatgaaag gttgggcttc ggaatcgttt tccgggacgc
    10021 cggctggatg atcctccagc gcggggatct catgctggag ttcttcgccc accccaactt
    10081 gtttattgca gcttataatg gttacaaata aagcaatagc atcacaaatt tcacaaataa
    10141 agcatttttt tcactgcatt ctagttgtgg tttgtccaaa ctcatcaatg tatcttatca
    10201 tgtctgtata ccgtcgacct ctagctagag cttggcgtaa tcatggtcat agctgtttcc
    10261 tgtgtgaaat tgttatccgc tcacaattcc acacaacata cgagccggaa gcataaagtg
    10321 taaagcctgg ggtgcctaat gagtgagcta actcacatta attgcgttgc gctcactgcc
    10381 cgctttccag tcgggaaacc tgtcgtgcca gctgcattaa tgaatcggcc aacgcgcggg
    10441 gagaggcggt ttgcgtattg ggcgctcttc cgcttcctcg ctcactgact cgctgcgctc
    10501 ggtcgttcgg ctgcggcgag cggtatcagc tcactcaaag gcggtaatac ggttatccac
    10561 agaatcaggg gataacgcag gaaagaacat gtgagcaaaa ggccagcaaa aggccaggaa
    10621 ccgtaaaaag gccgcgttgc tggcgttttt ccataggctc cgcccccctg acgagcatca
    10681 caaaaatcga cgctcaagtc agaggtggcg aaacccgaca ggactataaa gataccaggc
    10741 gtttccccct ggaagctccc tcgtgcgctc tcctgttccg accctgccgc ttaccggata
    10801 cctgtccgcc tttctccctt cgggaagcgt ggcgctttct catagctcac gctgtaggta
    10861 tctcagttcg gtgtaggtcg ttcgctccaa gctgggctgt gtgcacgaac cccccgttca
    10921 gcccgaccgc tgcgccttat ccggtaacta tcgtcttgag tccaacccgg taagacacga
    10981 cttatcgcca ctggcagcag ccactggtaa caggattagc agagcgaggt atgtaggcgg
    11041 tgctacagag ttcttgaagt ggtggcctaa ctacggctac actagaagaa cagtatttgg
    11101 tatctgcgct ctgctgaagc cagttacctt cggaaaaaga gttggtagct cttgatccgg
    11161 caaacaaacc accgctggta gcggtggttt ttttgtttgc aagcagcaga ttacgcgcag
    11221 aaaaaaagga tctcaagaag atcctttgat cttttctacg gggtctgacg ctcagtggaa
    11281 cgaaaactca cgttaaggga ttttggtcat gagattatca aaaaggatct tcacctagat
    11341 ccttttaaat taaaaatgaa gttttaaatc aatctaaagt atatatgagt aaacttggtc
    11401 tgacagttac caatgcttaa tcagtgaggc acctatctca gcgatctgtc tatttcgttc
    11461 atccatagtt gcctgactcc ccgtcgtgta gataactacg atacgggagg gcttaccatc
    11521 tggccccagt gctgcaatga taccgcgaga cccacgctca ccggctccag atttatcagc
    11581 aataaaccag ccagccggaa gggccgagcg cagaagtggt cctgcaactt tatccgcctc
    11641 catccagtct attaattgtt gccgggaagc tagagtaagt agttcgccag ttaatagttt
    11701 gcgcaacgtt gttgccattg ctacaggcat cgtggtgtca cgctcgtcgt ttggtatggc
    11761 ttcattcagc tccggttccc aacgatcaag gcgagttaca tgatccccca tgttgtgcaa
    11821 aaaagcggtt agctccttcg gtcctccgat cgttgtcaga agtaagttgg ccgcagtgtt
    11881 atcactcatg gttatggcag cactgcataa ttctcttact gtcatgccat ccgtaagatg
    11941 cttttctgtg actggtgagt actcaaccaa gtcattctga gaatagtgta tgcggcgacc
    12001 gagttgctct tgcccggcgt caatacggga taataccgcg ccacatagca gaactttaaa
    12061 agtgctcatc attggaaaac gttcttcggg gcgaaaactc tcaaggatct taccgctgtt
    12121 gagatccagt tcgatgtaac ccactcgtgc acccaactga tcttcagcat cttttacttt
    12181 caccagcgtt tctgggtgag caaaaacagg aaggcaaaat gccgcaaaaa agggaataag
    12241 ggcgacacgg aaatgttgaa tactcatact cttccttttt caatattatt gaagcattta
    12301 tcagggttat tgtctcatga gcggatacat atttgaatgt atttagaaaa ataaacaaat
    12361 aggggttccg cgcacatttc cccgaaaagt gccacctgac gtcgacggat cgggagatct
    12421 cccgatcccc tatggtgcac tctcagtaca atctgctctg atgccgcata gttaagccag
    12481 tatctgctcc ctgcttgtgt gttggaggtc gctgagtagt gcgcgagcaa aatttaagct
    12541 acaacaaggc aaggcttgac cgacaattgc atgaagaatc tgcttagggt taggcgtttt
    12601 gcgctgcttc gcgatgtacg ggccagatat acgcgttgac attgattatt gactagttat
    12661 taatagtaat caattacggg gtcattagtt catagcccat atatggagtt ccgcgttaca
    12721 taacttacgg taaatggccc gcctggctga ccgcccaacg acccccgccc attgacgtca
    12781 ataatgacgt atgttcccat agtaacgcca atagggactt tccattgacg tcaatgggtg
    12841 gagtatttac ggtaaactgc ccacttggca gtacatcaag tgtatcatat gccaagtacg
    12901 ccccctattg acgtcaatga cggtaaatgg cccgcctggc attatgccca gtacatgacc
    12961 ttatgggact ttcctacttg gcagtacatc tacgtattag tcatcgctat tac
//
