LOCUS       sequence-413638-       13013 bp    DNA     circular SYN 15-DEC-2025
DEFINITION  knock out NCKAP1 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-sgNCKAP1-1
FEATURES             Location/Qualifiers
     source          1..13013
                     /mol_type="other DNA"
                     /organism="synthetic DNA construct"
     promoter        5..203
                     /label=CMV promoter
                     /note="human cytomegalovirus (CMV) immediate early 
                     promoter"
     primer_bind     158..178
                     /label=CMV-F
                     /note="Human CMV immediate early promoter, forward primer"
     LTR             221..401
                     /label=5' LTR (truncated)
                     /note="truncated 5' long terminal repeat (LTR) from HIV-1"
     misc_feature    448..573
                     /label=HIV-1 Psi
                     /note="packaging signal of human immunodeficiency virus 
                     type 1"
     misc_feature    1066..1299
                     /label=RRE
                     /note="The Rev response element (RRE) of HIV-1 allows for 
                     Rev-dependent mRNA export from the nucleus to the 
                     cytoplasm."
     CDS             1484..1528
                     /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    1826..1943
                     /label=cPPT/CTS
                     /note="central polypurine tract and central termination 
                     sequence of HIV-1"
     promoter        1994..2242
                     /label=U6 promoter
                     /note="RNA polymerase III promoter for human U6 snRNA 
                     (Domitrovich & Kunkel, 2003)"
     primer_bind     1994..2014
                     /label=hU6-F
                     /note="Human U6 promoter, forward primer"
     primer_bind     2165..2184
                     /label=LKO.1 5'
                     /note="Human U6 promoter, forward primer"
     misc_RNA        2264..2339
                     /label=gRNA scaffold
                     /note="guide RNA scaffold for the Streptococcus pyogenes 
                     CRISPR/Cas9 system"
     promoter        2401..2612
                     /label=EF-1-alpha core promoter
                     /note="core promoter for human elongation factor 
                     EF-1-alpha"
     regulatory      2631..2640
                     /label=Kozak sequence
                     /note="vertebrate consensus sequence for strong initiation 
                     of translation (Kozak, 1987)"
                     /regulatory_class="other"
     CDS             2637..6740
                     /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             2637..6740
                     /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             6741..6788
                     /codon_start=1
                     /product="bipartite nuclear localization signal from 
                     nucleoplasmin"
                     /label=nucleoplasmin NLS
                     /translation="KRPAATKKAGQAKKKK"
     CDS             6789..6812
                     /codon_start=1
                     /product="FLAG(R) epitope tag, followed by an enterokinase 
                     cleavage site"
                     /label=FLAG
                     /translation="DYKDDDDK"
     CDS             6822..6878
                     /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             6879..7475
                     /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     7372..7392
                     /label=Puro-F
                     /note="Puromycin resistance gene, forward primer"
     misc_feature    7491..8079
                     /label=WPRE
                     /note="woodchuck hepatitis virus posttranscriptional 
                     regulatory element"
     primer_bind     complement(7544..7564)
                     /label=WPRE-R
                     /note="WPRE, reverse primer"
     CDS             complement(7962..7973)
                     /codon_start=1
                     /product="Factor Xa recognition and cleavage site"
                     /label=Factor Xa site
                     /translation="IEGR"
     LTR             8151..8384
                     /label=3' LTR (Delta-U3)
                     /note="self-inactivating 3' long terminal repeat (LTR) from
                     HIV-1"
     primer_bind     complement(8410..8427)
                     /label=BGH-rev
                     /note="Bovine growth hormone terminator, reverse primer. 
                     Also called BGH reverse"
     polyA_signal    8416..8640
                     /label=bGH poly(A) signal
                     /note="bovine growth hormone polyadenylation signal"
     rep_origin      8686..9114
                     /direction=RIGHT
                     /label=f1 ori
                     /note="f1 bacteriophage origin of replication; arrow 
                     indicates direction of (+) strand synthesis"
     primer_bind     complement(8773..8792)
                     /label=F1ori-R
                     /note="F1 origin, reverse primer"
     primer_bind     8983..9004
                     /label=F1ori-F
                     /note="F1 origin, forward primer"
     primer_bind     complement(9123..9143)
                     /label=pBABE 3'
                     /note="SV40 enhancer, reverse primer for pBABE vectors"
     promoter        9128..9457
                     /label=SV40 promoter
                     /note="SV40 enhancer and early promoter"
     rep_origin      9308..9443
                     /label=SV40 ori
                     /note="SV40 origin of replication"
     primer_bind     9370..9389
                     /label=SV40pro-F
                     /note="SV40 promoter/origin, forward primer"
     promoter        9505..9552
                     /label=EM7 promoter
                     /note="synthetic bacterial promoter "
     CDS             9571..9945
                     /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    10075..10196
                     /label=SV40 poly(A) signal
                     /note="SV40 polyadenylation signal"
     primer_bind     complement(10112..10131)
                     /label=SV40pA-R
                     /note="SV40 polyA, reverse primer"
     primer_bind     10166..10185
                     /label=EBV-rev
                     /note="SV40 polyA terminator, reverse primer"
     primer_bind     complement(10245..10261)
                     /label=M13 rev
                     /note="common sequencing primer, one of multiple similar 
                     variants"
     primer_bind     complement(10245..10261)
                     /label=M13 Reverse
                     /note="In lacZ gene. Also called M13-rev"
     primer_bind     complement(10258..10280)
                     /label=M13/pUC Reverse
                     /note="In lacZ gene"
     protein_bind    10269..10285
                     /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(10293..10299,10300..10317,10318..10323))
                     /label=lac promoter
                     /note="promoter for the E. coli lac operon"
     protein_bind    10338..10359
                     /label=CAP binding site
                     /bound_moiety="E. coli catabolite activator protein"
                     /note="CAP binding activates transcription in the presence 
                     of cAMP."
     primer_bind     complement(10476..10493)
                     /label=L4440
                     /note="L4440 vector, forward primer"
     rep_origin      complement(10647..11235)
                     /direction=LEFT
                     /label=ori
                     /note="high-copy-number ColE1/pMB1/pBR322/pUC origin of 
                     replication"
     primer_bind     complement(10727..10746)
                     /label=pBR322ori-F
                     /note="pBR322 origin, forward primer"
     CDS             complement(join(11406..12197,12198..12266))
                     /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     12029..12048
                     /label=Amp-R
                     /note="Ampicillin resistance gene, reverse primer"
     promoter        complement(12267..12371)
                     /gene="bla"
                     /label=AmpR promoter
     primer_bind     complement(12446..12465)
                     /label=pRS-marker
                     /note="pRS vectors, use to sequence yeast selectable 
                     marker"
     enhancer        join(12637..13013,1..3)
                     /label=CMV enhancer
                     /note="human cytomegalovirus immediate early enhancer"
ORIGIN
        1 atggtgatgc ggttttggca gtacatcaat gggcgtggat agcggtttga ctcacgggga
       61 tttccaagtc tccaccccat tgacgtcaat gggagtttgt tttggcacca aaatcaacgg
      121 gactttccaa aatgtcgtaa caactccgcc ccattgacgc aaatgggcgg taggcgtgta
      181 cggtgggagg tctatataag cagcgcgttt tgcctgtact gggtctctct ggttagacca
      241 gatctgagcc tgggagctct ctggctaact agggaaccca ctgcttaagc ctcaataaag
      301 cttgccttga gtgcttcaag tagtgtgtgc ccgtctgttg tgtgactctg gtaactagag
      361 atccctcaga cccttttagt cagtgtggaa aatctctagc agtggcgccc gaacagggac
      421 ttgaaagcga aagggaaacc agaggagctc tctcgacgca ggactcggct tgctgaagcg
      481 cgcacggcaa gaggcgaggg gcggcgactg gtgagtacgc caaaaatttt gactagcgga
      541 ggctagaagg agagagatgg gtgcgagagc gtcagtatta agcgggggag aattagatcg
      601 cgatgggaaa aaattcggtt aaggccaggg ggaaagaaaa aatataaatt aaaacatata
      661 gtatgggcaa gcagggagct agaacgattc gcagttaatc ctggcctgtt agaaacatca
      721 gaaggctgta gacaaatact gggacagcta caaccatccc ttcagacagg atcagaagaa
      781 cttagatcat tatataatac agtagcaacc ctctattgtg tgcatcaaag gatagagata
      841 aaagacacca aggaagcttt agacaagata gaggaagagc aaaacaaaag taagaccacc
      901 gcacagcaag cggccgctga tcttcagacc tggaggagga gatatgaggg acaattggag
      961 aagtgaatta tataaatata aagtagtaaa aattgaacca ttaggagtag cacccaccaa
     1021 ggcaaagaga agagtggtgc agagagaaaa aagagcagtg ggaataggag ctttgttcct
     1081 tgggttcttg ggagcagcag gaagcactat gggcgcagcg tcaatgacgc tgacggtaca
     1141 ggccagacaa ttattgtctg gtatagtgca gcagcagaac aatttgctga gggctattga
     1201 ggcgcaacag catctgttgc aactcacagt ctggggcatc aagcagctcc aggcaagaat
     1261 cctggctgtg gaaagatacc taaaggatca acagctcctg gggatttggg gttgctctgg
     1321 aaaactcatt tgcaccactg ctgtgccttg gaatgctagt tggagtaata aatctctgga
     1381 acagatttgg aatcacacga cctggatgga gtgggacaga gaaattaaca attacacaag
     1441 cttaatacac tccttaattg aagaatcgca aaaccagcaa gaaaagaatg aacaagaatt
     1501 attggaatta gataaatggg caagtttgtg gaattggttt aacataacaa attggctgtg
     1561 gtatataaaa ttattcataa tgatagtagg aggcttggta ggtttaagaa tagtttttgc
     1621 tgtactttct atagtgaata gagttaggca gggatattca ccattatcgt ttcagaccca
     1681 cctcccaacc ccgaggggac ccgacaggcc cgaaggaata gaagaagaag gtggagagag
     1741 agacagagac agatccattc gattagtgaa cggatcggca ctgcgtgcgc caattctgca
     1801 gacaaatggc agtattcatc cacaatttta aaagaaaagg ggggattggg gggtacagtg
     1861 caggggaaag aatagtagac ataatagcaa cagacataca aactaaagaa ttacaaaaac
     1921 aaattacaaa aattcaaaat tttcgggttt attacaggga cagcagagat ccagtttggt
     1981 taattaaggt accgagggcc tatttcccat gattccttca tatttgcata tacgatacaa
     2041 ggctgttaga gagataatta gaattaattt gactgtaaac acaaagatat tagtacaaaa
     2101 tacgtgacgt agaaagtaat aatttcttgg gtagtttgca gttttaaaat tatgttttaa
     2161 aatggactat catatgctta ccgtaacttg aaagtatttc gatttcttgg ctttatatat
     2221 cttgtggaaa ggacgaaaca ccgctcacca tcctcaacga ccggttttag agctagaaat
     2281 agcaagttaa aataaggcta gtccgttatc aacttgaaaa agtggcaccg agtcggtgct
     2341 tttttgaatt cgctagctag gtcttgaaag gagtgggaat tggctccggt gcccgtcagt
     2401 gggcagagcg cacatcgccc acagtccccg agaagttggg gggaggggtc ggcaattgat
     2461 ccggtgccta gagaaggtgg cgcggggtaa actgggaaag tgatgtcgtg tactggctcc
     2521 gcctttttcc cgagggtggg ggagaaccgt atataagtgc agtagtcgcc gtgaacgttc
     2581 tttttcgcaa cgggtttgcc gccagaacac aggaccggtt ctagagcgct gccaccatgg
     2641 acaagaagta cagcatcggc ctggacatcg gcaccaactc tgtgggctgg gccgtgatca
     2701 ccgacgagta caaggtgccc agcaagaaat tcaaggtgct gggcaacacc gaccggcaca
     2761 gcatcaagaa gaacctgatc ggagccctgc tgttcgacag cggcgaaaca gccgaggcca
     2821 cccggctgaa gagaaccgcc agaagaagat acaccagacg gaagaaccgg atctgctatc
     2881 tgcaagagat cttcagcaac gagatggcca aggtggacga cagcttcttc cacagactgg
     2941 aagagtcctt cctggtggaa gaggataaga agcacgagcg gcaccccatc ttcggcaaca
     3001 tcgtggacga ggtggcctac cacgagaagt accccaccat ctaccacctg agaaagaaac
     3061 tggtggacag caccgacaag gccgacctgc ggctgatcta tctggccctg gcccacatga
     3121 tcaagttccg gggccacttc ctgatcgagg gcgacctgaa ccccgacaac agcgacgtgg
     3181 acaagctgtt catccagctg gtgcagacct acaaccagct gttcgaggaa aaccccatca
     3241 acgccagcgg cgtggacgcc aaggccatcc tgtctgccag actgagcaag agcagacggc
     3301 tggaaaatct gatcgcccag ctgcccggcg agaagaagaa tggcctgttc ggaaacctga
     3361 ttgccctgag cctgggcctg acccccaact tcaagagcaa cttcgacctg gccgaggatg
     3421 ccaaactgca gctgagcaag gacacctacg acgacgacct ggacaacctg ctggcccaga
     3481 tcggcgacca gtacgccgac ctgtttctgg ccgccaagaa cctgtccgac gccatcctgc
     3541 tgagcgacat cctgagagtg aacaccgaga tcaccaaggc ccccctgagc gcctctatga
     3601 tcaagagata cgacgagcac caccaggacc tgaccctgct gaaagctctc gtgcggcagc
     3661 agctgcctga gaagtacaaa gagattttct tcgaccagag caagaacggc tacgccggct
     3721 acattgacgg cggagccagc caggaagagt tctacaagtt catcaagccc atcctggaaa
     3781 agatggacgg caccgaggaa ctgctcgtga agctgaacag agaggacctg ctgcggaagc
     3841 agcggacctt cgacaacggc agcatccccc accagatcca cctgggagag ctgcacgcca
     3901 ttctgcggcg gcaggaagat ttttacccat tcctgaagga caaccgggaa aagatcgaga
     3961 agatcctgac cttccgcatc ccctactacg tgggccctct ggccagggga aacagcagat
     4021 tcgcctggat gaccagaaag agcgaggaaa ccatcacccc ctggaacttc gaggaagtgg
     4081 tggacaaggg cgcttccgcc cagagcttca tcgagcggat gaccaacttc gataagaacc
     4141 tgcccaacga gaaggtgctg cccaagcaca gcctgctgta cgagtacttc accgtgtata
     4201 acgagctgac caaagtgaaa tacgtgaccg agggaatgag aaagcccgcc ttcctgagcg
     4261 gcgagcagaa aaaggccatc gtggacctgc tgttcaagac caaccggaaa gtgaccgtga
     4321 agcagctgaa agaggactac ttcaagaaaa tcgagtgctt cgactccgtg gaaatctccg
     4381 gcgtggaaga tcggttcaac gcctccctgg gcacatacca cgatctgctg aaaattatca
     4441 aggacaagga cttcctggac aatgaggaaa acgaggacat tctggaagat atcgtgctga
     4501 ccctgacact gtttgaggac agagagatga tcgaggaacg gctgaaaacc tatgcccacc
     4561 tgttcgacga caaagtgatg aagcagctga agcggcggag atacaccggc tggggcaggc
     4621 tgagccggaa gctgatcaac ggcatccggg acaagcagtc cggcaagaca atcctggatt
     4681 tcctgaagtc cgacggcttc gccaacagaa acttcatgca gctgatccac gacgacagcc
     4741 tgacctttaa agaggacatc cagaaagccc aggtgtccgg ccagggcgat agcctgcacg
     4801 agcacattgc caatctggcc ggcagccccg ccattaagaa gggcatcctg cagacagtga
     4861 aggtggtgga cgagctcgtg aaagtgatgg gccggcacaa gcccgagaac atcgtgatcg
     4921 aaatggccag agagaaccag accacccaga agggacagaa gaacagccgc gagagaatga
     4981 agcggatcga agagggcatc aaagagctgg gcagccagat cctgaaagaa caccccgtgg
     5041 aaaacaccca gctgcagaac gagaagctgt acctgtacta cctgcagaat gggcgggata
     5101 tgtacgtgga ccaggaactg gacatcaacc ggctgtccga ctacgatgtg gaccatatcg
     5161 tgcctcagag ctttctgaag gacgactcca tcgacaacaa ggtgctgacc agaagcgaca
     5221 agaaccgggg caagagcgac aacgtgccct ccgaagaggt cgtgaagaag atgaagaact
     5281 actggcggca gctgctgaac gccaagctga ttacccagag aaagttcgac aatctgacca
     5341 aggccgagag aggcggcctg agcgaactgg ataaggccgg cttcatcaag agacagctgg
     5401 tggaaacccg gcagatcaca aagcacgtgg cacagatcct ggactcccgg atgaacacta
     5461 agtacgacga gaatgacaag ctgatccggg aagtgaaagt gatcaccctg aagtccaagc
     5521 tggtgtccga tttccggaag gatttccagt tttacaaagt gcgcgagatc aacaactacc
     5581 accacgccca cgacgcctac ctgaacgccg tcgtgggaac cgccctgatc aaaaagtacc
     5641 ctaagctgga aagcgagttc gtgtacggcg actacaaggt gtacgacgtg cggaagatga
     5701 tcgccaagag cgagcaggaa atcggcaagg ctaccgccaa gtacttcttc tacagcaaca
     5761 tcatgaactt tttcaagacc gagattaccc tggccaacgg cgagatccgg aagcggcctc
     5821 tgatcgagac aaacggcgaa accggggaga tcgtgtggga taagggccgg gattttgcca
     5881 ccgtgcggaa agtgctgagc atgccccaag tgaatatcgt gaaaaagacc gaggtgcaga
     5941 caggcggctt cagcaaagag tctatcctgc ccaagaggaa cagcgataag ctgatcgcca
     6001 gaaagaagga ctgggaccct aagaagtacg gcggcttcga cagccccacc gtggcctatt
     6061 ctgtgctggt ggtggccaaa gtggaaaagg gcaagtccaa gaaactgaag agtgtgaaag
     6121 agctgctggg gatcaccatc atggaaagaa gcagcttcga gaagaatccc atcgactttc
     6181 tggaagccaa gggctacaaa gaagtgaaaa aggacctgat catcaagctg cctaagtact
     6241 ccctgttcga gctggaaaac ggccggaaga gaatgctggc ctctgccggc gaactgcaga
     6301 agggaaacga actggccctg ccctccaaat atgtgaactt cctgtacctg gccagccact
     6361 atgagaagct gaagggctcc cccgaggata atgagcagaa acagctgttt gtggaacagc
     6421 acaagcacta cctggacgag atcatcgagc agatcagcga gttctccaag agagtgatcc
     6481 tggccgacgc taatctggac aaagtgctgt ccgcctacaa caagcaccgg gataagccca
     6541 tcagagagca ggccgagaat atcatccacc tgtttaccct gaccaatctg ggagcccctg
     6601 ccgccttcaa gtactttgac accaccatcg accggaagag gtacaccagc accaaagagg
     6661 tgctggacgc caccctgatc caccagagca tcaccggcct gtacgagaca cggatcgacc
     6721 tgtctcagct gggaggcgac aagcgacctg ccgccacaaa gaaggctgga caggctaaga
     6781 agaagaaaga ttacaaagac gatgacgata agggatccgg cgcaacaaac ttctctctgc
     6841 tgaaacaagc cggagatgtc gaagagaatc ctggaccgac cgagtacaag cccacggtgc
     6901 gcctcgccac ccgcgacgac gtccccaggg ccgtacgcac cctcgccgcc gcgttcgccg
     6961 actaccccgc cacgcgccac accgtcgatc cggaccgcca catcgagcgg gtcaccgagc
     7021 tgcaagaact cttcctcacg cgcgtcgggc tcgacatcgg caaggtgtgg gtcgcggacg
     7081 acggcgccgc ggtggcggtc tggaccacgc cggagagcgt cgaagcgggg gcggtgttcg
     7141 ccgagatcgg cccgcgcatg gccgagttga gcggttcccg gctggccgcg cagcaacaga
     7201 tggaaggcct cctggcgccg caccggccca aggagcccgc gtggttcctg gccaccgtcg
     7261 gagtctcgcc cgaccaccag ggcaagggtc tgggcagcgc cgtcgtgctc cccggagtgg
     7321 aggcggccga gcgcgccggg gtgcccgcct tcctggagac ctccgcgccc cgcaacctcc
     7381 ccttctacga gcggctcggc ttcaccgtca ccgccgacgt cgaggtgccc gaaggaccgc
     7441 gcacctggtg catgacccgc aagcccggtg cctgaacgcg ttaagtcgac aatcaacctc
     7501 tggattacaa aatttgtgaa agattgactg gtattcttaa ctatgttgct ccttttacgc
     7561 tatgtggata cgctgcttta atgcctttgt atcatgctat tgcttcccgt atggctttca
     7621 ttttctcctc cttgtataaa tcctggttgc tgtctcttta tgaggagttg tggcccgttg
     7681 tcaggcaacg tggcgtggtg tgcactgtgt ttgctgacgc aacccccact ggttggggca
     7741 ttgccaccac ctgtcagctc ctttccggga ctttcgcttt ccccctccct attgccacgg
     7801 cggaactcat cgccgcctgc cttgcccgct gctggacagg ggctcggctg ttgggcactg
     7861 acaattccgt ggtgttgtcg gggaaatcat cgtcctttcc ttggctgctc gcctgtgttg
     7921 ccacctggat tctgcgcggg acgtccttct gctacgtccc ttcggccctc aatccagcgg
     7981 accttccttc ccgcggcctg ctgccggctc tgcggcctct tccgcgtctt cgccttcgcc
     8041 ctcagacgag tcggatctcc ctttgggccg cctccccgcg tcgactttaa gaccaatgac
     8101 ttacaaggca gctgtagatc ttagccactt tttaaaagaa aaggggggac tggaagggct
     8161 aattcactcc caacgaagac aagatctgct ttttgcttgt actgggtctc tctggttaga
     8221 ccagatctga gcctgggagc tctctggcta actagggaac ccactgctta agcctcaata
     8281 aagcttgcct tgagtgcttc aagtagtgtg tgcccgtctg ttgtgtgact ctggtaacta
     8341 gagatccctc agaccctttt agtcagtgtg gaaaatctct agcagggccc gtttaaaccc
     8401 gctgatcagc ctcgactgtg ccttctagtt gccagccatc tgttgtttgc ccctcccccg
     8461 tgccttcctt gaccctggaa ggtgccactc ccactgtcct ttcctaataa aatgaggaaa
     8521 ttgcatcgca ttgtctgagt aggtgtcatt ctattctggg gggtggggtg gggcaggaca
     8581 gcaaggggga ggattgggaa gacaatagca ggcatgctgg ggatgcggtg ggctctatgg
     8641 cttctgaggc ggaaagaacc agctggggct ctagggggta tccccacgcg ccctgtagcg
     8701 gcgcattaag cgcggcgggt gtggtggtta cgcgcagcgt gaccgctaca cttgccagcg
     8761 ccctagcgcc cgctcctttc gctttcttcc cttcctttct cgccacgttc gccggctttc
     8821 cccgtcaagc tctaaatcgg gggctccctt tagggttccg atttagtgct ttacggcacc
     8881 tcgaccccaa aaaacttgat tagggtgatg gttcacgtag tgggccatcg ccctgataga
     8941 cggtttttcg ccctttgacg ttggagtcca cgttctttaa tagtggactc ttgttccaaa
     9001 ctggaacaac actcaaccct atctcggtct attcttttga tttataaggg attttgccga
     9061 tttcggccta ttggttaaaa aatgagctga tttaacaaaa atttaacgcg aattaattct
     9121 gtggaatgtg tgtcagttag ggtgtggaaa gtccccaggc tccccagcag gcagaagtat
     9181 gcaaagcatg catctcaatt agtcagcaac caggtgtgga aagtccccag gctccccagc
     9241 aggcagaagt atgcaaagca tgcatctcaa ttagtcagca accatagtcc cgcccctaac
     9301 tccgcccatc ccgcccctaa ctccgcccag ttccgcccat tctccgcccc atggctgact
     9361 aatttttttt atttatgcag aggccgaggc cgcctctgcc tctgagctat tccagaagta
     9421 gtgaggaggc ttttttggag gcctaggctt ttgcaaaaag ctcccgggag cttgtatatc
     9481 cattttcgga tctgatcagc acgtgttgac aattaatcat cggcatagta tatcggcata
     9541 gtataatacg acaaggtgag gaactaaacc atggccaagt tgaccagtgc cgttccggtg
     9601 ctcaccgcgc gcgacgtcgc cggagcggtc gagttctgga ccgaccggct cgggttctcc
     9661 cgggacttcg tggaggacga cttcgccggt gtggtccggg acgacgtgac cctgttcatc
     9721 agcgcggtcc aggaccaggt ggtgccggac aacaccctgg cctgggtgtg ggtgcgcggc
     9781 ctggacgagc tgtacgccga gtggtcggag gtcgtgtcca cgaacttccg ggacgcctcc
     9841 gggccggcca tgaccgagat cggcgagcag ccgtgggggc gggagttcgc cctgcgcgac
     9901 ccggccggca actgcgtgca cttcgtggcc gaggagcagg actgacacgt gctacgagat
     9961 ttcgattcca ccgccgcctt ctatgaaagg ttgggcttcg gaatcgtttt ccgggacgcc
    10021 ggctggatga tcctccagcg cggggatctc atgctggagt tcttcgccca ccccaacttg
    10081 tttattgcag cttataatgg ttacaaataa agcaatagca tcacaaattt cacaaataaa
    10141 gcattttttt cactgcattc tagttgtggt ttgtccaaac tcatcaatgt atcttatcat
    10201 gtctgtatac cgtcgacctc tagctagagc ttggcgtaat catggtcata gctgtttcct
    10261 gtgtgaaatt gttatccgct cacaattcca cacaacatac gagccggaag cataaagtgt
    10321 aaagcctggg gtgcctaatg agtgagctaa ctcacattaa ttgcgttgcg ctcactgccc
    10381 gctttccagt cgggaaacct gtcgtgccag ctgcattaat gaatcggcca acgcgcgggg
    10441 agaggcggtt tgcgtattgg gcgctcttcc gcttcctcgc tcactgactc gctgcgctcg
    10501 gtcgttcggc tgcggcgagc ggtatcagct cactcaaagg cggtaatacg gttatccaca
    10561 gaatcagggg ataacgcagg aaagaacatg tgagcaaaag gccagcaaaa ggccaggaac
    10621 cgtaaaaagg ccgcgttgct ggcgtttttc cataggctcc gcccccctga cgagcatcac
    10681 aaaaatcgac gctcaagtca gaggtggcga aacccgacag gactataaag ataccaggcg
    10741 tttccccctg gaagctccct cgtgcgctct cctgttccga ccctgccgct taccggatac
    10801 ctgtccgcct ttctcccttc gggaagcgtg gcgctttctc atagctcacg ctgtaggtat
    10861 ctcagttcgg tgtaggtcgt tcgctccaag ctgggctgtg tgcacgaacc ccccgttcag
    10921 cccgaccgct gcgccttatc cggtaactat cgtcttgagt ccaacccggt aagacacgac
    10981 ttatcgccac tggcagcagc cactggtaac aggattagca gagcgaggta tgtaggcggt
    11041 gctacagagt tcttgaagtg gtggcctaac tacggctaca ctagaagaac agtatttggt
    11101 atctgcgctc tgctgaagcc agttaccttc ggaaaaagag ttggtagctc ttgatccggc
    11161 aaacaaacca ccgctggtag cggtggtttt tttgtttgca agcagcagat tacgcgcaga
    11221 aaaaaaggat ctcaagaaga tcctttgatc ttttctacgg ggtctgacgc tcagtggaac
    11281 gaaaactcac gttaagggat tttggtcatg agattatcaa aaaggatctt cacctagatc
    11341 cttttaaatt aaaaatgaag ttttaaatca atctaaagta tatatgagta aacttggtct
    11401 gacagttacc aatgcttaat cagtgaggca cctatctcag cgatctgtct atttcgttca
    11461 tccatagttg cctgactccc cgtcgtgtag ataactacga tacgggaggg cttaccatct
    11521 ggccccagtg ctgcaatgat accgcgagac ccacgctcac cggctccaga tttatcagca
    11581 ataaaccagc cagccggaag ggccgagcgc agaagtggtc ctgcaacttt atccgcctcc
    11641 atccagtcta ttaattgttg ccgggaagct agagtaagta gttcgccagt taatagtttg
    11701 cgcaacgttg ttgccattgc tacaggcatc gtggtgtcac gctcgtcgtt tggtatggct
    11761 tcattcagct ccggttccca acgatcaagg cgagttacat gatcccccat gttgtgcaaa
    11821 aaagcggtta gctccttcgg tcctccgatc gttgtcagaa gtaagttggc cgcagtgtta
    11881 tcactcatgg ttatggcagc actgcataat tctcttactg tcatgccatc cgtaagatgc
    11941 ttttctgtga ctggtgagta ctcaaccaag tcattctgag aatagtgtat gcggcgaccg
    12001 agttgctctt gcccggcgtc aatacgggat aataccgcgc cacatagcag aactttaaaa
    12061 gtgctcatca ttggaaaacg ttcttcgggg cgaaaactct caaggatctt accgctgttg
    12121 agatccagtt cgatgtaacc cactcgtgca cccaactgat cttcagcatc ttttactttc
    12181 accagcgttt ctgggtgagc aaaaacagga aggcaaaatg ccgcaaaaaa gggaataagg
    12241 gcgacacgga aatgttgaat actcatactc ttcctttttc aatattattg aagcatttat
    12301 cagggttatt gtctcatgag cggatacata tttgaatgta tttagaaaaa taaacaaata
    12361 ggggttccgc gcacatttcc ccgaaaagtg ccacctgacg tcgacggatc gggagatctc
    12421 ccgatcccct atggtgcact ctcagtacaa tctgctctga tgccgcatag ttaagccagt
    12481 atctgctccc tgcttgtgtg ttggaggtcg ctgagtagtg cgcgagcaaa atttaagcta
    12541 caacaaggca aggcttgacc gacaattgca tgaagaatct gcttagggtt aggcgttttg
    12601 cgctgcttcg cgatgtacgg gccagatata cgcgttgaca ttgattattg actagttatt
    12661 aatagtaatc aattacgggg tcattagttc atagcccata tatggagttc cgcgttacat
    12721 aacttacggt aaatggcccg cctggctgac cgcccaacga cccccgccca ttgacgtcaa
    12781 taatgacgta tgttcccata gtaacgccaa tagggacttt ccattgacgt caatgggtgg
    12841 agtatttacg gtaaactgcc cacttggcag tacatcaagt gtatcatatg ccaagtacgc
    12901 cccctattga cgtcaatgac ggtaaatggc ccgcctggca ttatgcccag tacatgacct
    12961 tatgggactt tcctacttgg cagtacatct acgtattagt catcgctatt acc
//
