LOCUS       sequence-413639-       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-sgNCKAP1-2
FEATURES             Location/Qualifiers
     source          1..13013
                     /mol_type="other DNA"
                     /organism="synthetic DNA construct"
     primer_bind     144..164
                     /label=CMV-F
                     /note="Human CMV immediate early promoter, forward primer"
     LTR             207..387
                     /label=5' LTR (truncated)
                     /note="truncated 5' long terminal repeat (LTR) from HIV-1"
     misc_feature    434..559
                     /label=HIV-1 Psi
                     /note="packaging signal of human immunodeficiency virus 
                     type 1"
     misc_feature    1052..1285
                     /label=RRE
                     /note="The Rev response element (RRE) of HIV-1 allows for 
                     Rev-dependent mRNA export from the nucleus to the 
                     cytoplasm."
     CDS             1470..1514
                     /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    1812..1929
                     /label=cPPT/CTS
                     /note="central polypurine tract and central termination 
                     sequence of HIV-1"
     promoter        1980..2228
                     /label=U6 promoter
                     /note="RNA polymerase III promoter for human U6 snRNA 
                     (Domitrovich & Kunkel, 2003)"
     primer_bind     1980..2000
                     /label=hU6-F
                     /note="Human U6 promoter, forward primer"
     primer_bind     2151..2170
                     /label=LKO.1 5'
                     /note="Human U6 promoter, forward primer"
     misc_RNA        2250..2325
                     /label=gRNA scaffold
                     /note="guide RNA scaffold for the Streptococcus pyogenes 
                     CRISPR/Cas9 system"
     promoter        2387..2598
                     /label=EF-1-alpha core promoter
                     /note="core promoter for human elongation factor 
                     EF-1-alpha"
     regulatory      2617..2626
                     /label=Kozak sequence
                     /note="vertebrate consensus sequence for strong initiation 
                     of translation (Kozak, 1987)"
                     /regulatory_class="other"
     CDS             2623..6726
                     /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             2623..6726
                     /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             6727..6774
                     /codon_start=1
                     /product="bipartite nuclear localization signal from 
                     nucleoplasmin"
                     /label=nucleoplasmin NLS
                     /translation="KRPAATKKAGQAKKKK"
     CDS             6775..6798
                     /codon_start=1
                     /product="FLAG(R) epitope tag, followed by an enterokinase 
                     cleavage site"
                     /label=FLAG
                     /translation="DYKDDDDK"
     CDS             6808..6864
                     /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             6865..7461
                     /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     7358..7378
                     /label=Puro-F
                     /note="Puromycin resistance gene, forward primer"
     misc_feature    7477..8065
                     /label=WPRE
                     /note="woodchuck hepatitis virus posttranscriptional 
                     regulatory element"
     primer_bind     complement(7530..7550)
                     /label=WPRE-R
                     /note="WPRE, reverse primer"
     CDS             complement(7948..7959)
                     /codon_start=1
                     /product="Factor Xa recognition and cleavage site"
                     /label=Factor Xa site
                     /translation="IEGR"
     LTR             8137..8370
                     /label=3' LTR (Delta-U3)
                     /note="self-inactivating 3' long terminal repeat (LTR) from
                     HIV-1"
     primer_bind     complement(8396..8413)
                     /label=BGH-rev
                     /note="Bovine growth hormone terminator, reverse primer. 
                     Also called BGH reverse"
     polyA_signal    8402..8626
                     /label=bGH poly(A) signal
                     /note="bovine growth hormone polyadenylation signal"
     rep_origin      8672..9100
                     /direction=RIGHT
                     /label=f1 ori
                     /note="f1 bacteriophage origin of replication; arrow 
                     indicates direction of (+) strand synthesis"
     primer_bind     complement(8759..8778)
                     /label=F1ori-R
                     /note="F1 origin, reverse primer"
     primer_bind     8969..8990
                     /label=F1ori-F
                     /note="F1 origin, forward primer"
     primer_bind     complement(9109..9129)
                     /label=pBABE 3'
                     /note="SV40 enhancer, reverse primer for pBABE vectors"
     promoter        9114..9443
                     /label=SV40 promoter
                     /note="SV40 enhancer and early promoter"
     rep_origin      9294..9429
                     /label=SV40 ori
                     /note="SV40 origin of replication"
     primer_bind     9356..9375
                     /label=SV40pro-F
                     /note="SV40 promoter/origin, forward primer"
     promoter        9491..9538
                     /label=EM7 promoter
                     /note="synthetic bacterial promoter "
     CDS             9557..9931
                     /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    10061..10182
                     /label=SV40 poly(A) signal
                     /note="SV40 polyadenylation signal"
     primer_bind     complement(10098..10117)
                     /label=SV40pA-R
                     /note="SV40 polyA, reverse primer"
     primer_bind     10152..10171
                     /label=EBV-rev
                     /note="SV40 polyA terminator, reverse primer"
     primer_bind     complement(10231..10247)
                     /label=M13 rev
                     /note="common sequencing primer, one of multiple similar 
                     variants"
     primer_bind     complement(10231..10247)
                     /label=M13 Reverse
                     /note="In lacZ gene. Also called M13-rev"
     primer_bind     complement(10244..10266)
                     /label=M13/pUC Reverse
                     /note="In lacZ gene"
     protein_bind    10255..10271
                     /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(10279..10285,10286..10303,10304..10309))
                     /label=lac promoter
                     /note="promoter for the E. coli lac operon"
     protein_bind    10324..10345
                     /label=CAP binding site
                     /bound_moiety="E. coli catabolite activator protein"
                     /note="CAP binding activates transcription in the presence 
                     of cAMP."
     primer_bind     complement(10462..10479)
                     /label=L4440
                     /note="L4440 vector, forward primer"
     rep_origin      complement(10633..11221)
                     /direction=LEFT
                     /label=ori
                     /note="high-copy-number ColE1/pMB1/pBR322/pUC origin of 
                     replication"
     primer_bind     complement(10713..10732)
                     /label=pBR322ori-F
                     /note="pBR322 origin, forward primer"
     CDS             complement(join(11392..12183,12184..12252))
                     /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     12015..12034
                     /label=Amp-R
                     /note="Ampicillin resistance gene, reverse primer"
     promoter        complement(12253..12357)
                     /gene="bla"
                     /label=AmpR promoter
     primer_bind     complement(12432..12451)
                     /label=pRS-marker
                     /note="pRS vectors, use to sequence yeast selectable 
                     marker"
     enhancer        12623..13002
                     /label=CMV enhancer
                     /note="human cytomegalovirus immediate early enhancer"
     promoter        join(13004..13013,1..189)
                     /label=CMV promoter
                     /note="human cytomegalovirus (CMV) immediate early 
                     promoter"
ORIGIN
        1 ttggcagtac atcaatgggc gtggatagcg gtttgactca cggggatttc caagtctcca
       61 ccccattgac gtcaatggga gtttgttttg gcaccaaaat caacgggact ttccaaaatg
      121 tcgtaacaac tccgccccat tgacgcaaat gggcggtagg cgtgtacggt gggaggtcta
      181 tataagcagc gcgttttgcc tgtactgggt ctctctggtt agaccagatc tgagcctggg
      241 agctctctgg ctaactaggg aacccactgc ttaagcctca ataaagcttg ccttgagtgc
      301 ttcaagtagt gtgtgcccgt ctgttgtgtg actctggtaa ctagagatcc ctcagaccct
      361 tttagtcagt gtggaaaatc tctagcagtg gcgcccgaac agggacttga aagcgaaagg
      421 gaaaccagag gagctctctc gacgcaggac tcggcttgct gaagcgcgca cggcaagagg
      481 cgaggggcgg cgactggtga gtacgccaaa aattttgact agcggaggct agaaggagag
      541 agatgggtgc gagagcgtca gtattaagcg ggggagaatt agatcgcgat gggaaaaaat
      601 tcggttaagg ccagggggaa agaaaaaata taaattaaaa catatagtat gggcaagcag
      661 ggagctagaa cgattcgcag ttaatcctgg cctgttagaa acatcagaag gctgtagaca
      721 aatactggga cagctacaac catcccttca gacaggatca gaagaactta gatcattata
      781 taatacagta gcaaccctct attgtgtgca tcaaaggata gagataaaag acaccaagga
      841 agctttagac aagatagagg aagagcaaaa caaaagtaag accaccgcac agcaagcggc
      901 cgctgatctt cagacctgga ggaggagata tgagggacaa ttggagaagt gaattatata
      961 aatataaagt agtaaaaatt gaaccattag gagtagcacc caccaaggca aagagaagag
     1021 tggtgcagag agaaaaaaga gcagtgggaa taggagcttt gttccttggg ttcttgggag
     1081 cagcaggaag cactatgggc gcagcgtcaa tgacgctgac ggtacaggcc agacaattat
     1141 tgtctggtat agtgcagcag cagaacaatt tgctgagggc tattgaggcg caacagcatc
     1201 tgttgcaact cacagtctgg ggcatcaagc agctccaggc aagaatcctg gctgtggaaa
     1261 gatacctaaa ggatcaacag ctcctgggga tttggggttg ctctggaaaa ctcatttgca
     1321 ccactgctgt gccttggaat gctagttgga gtaataaatc tctggaacag atttggaatc
     1381 acacgacctg gatggagtgg gacagagaaa ttaacaatta cacaagctta atacactcct
     1441 taattgaaga atcgcaaaac cagcaagaaa agaatgaaca agaattattg gaattagata
     1501 aatgggcaag tttgtggaat tggtttaaca taacaaattg gctgtggtat ataaaattat
     1561 tcataatgat agtaggaggc ttggtaggtt taagaatagt ttttgctgta ctttctatag
     1621 tgaatagagt taggcaggga tattcaccat tatcgtttca gacccacctc ccaaccccga
     1681 ggggacccga caggcccgaa ggaatagaag aagaaggtgg agagagagac agagacagat
     1741 ccattcgatt agtgaacgga tcggcactgc gtgcgccaat tctgcagaca aatggcagta
     1801 ttcatccaca attttaaaag aaaagggggg attggggggt acagtgcagg ggaaagaata
     1861 gtagacataa tagcaacaga catacaaact aaagaattac aaaaacaaat tacaaaaatt
     1921 caaaattttc gggtttatta cagggacagc agagatccag tttggttaat taaggtaccg
     1981 agggcctatt tcccatgatt ccttcatatt tgcatatacg atacaaggct gttagagaga
     2041 taattagaat taatttgact gtaaacacaa agatattagt acaaaatacg tgacgtagaa
     2101 agtaataatt tcttgggtag tttgcagttt taaaattatg ttttaaaatg gactatcata
     2161 tgcttaccgt aacttgaaag tatttcgatt tcttggcttt atatatcttg tggaaaggac
     2221 gaaacaccgt gacataagag aatgcaaggg ttttagagct agaaatagca agttaaaata
     2281 aggctagtcc gttatcaact tgaaaaagtg gcaccgagtc ggtgcttttt tgaattcgct
     2341 agctaggtct tgaaaggagt gggaattggc tccggtgccc gtcagtgggc agagcgcaca
     2401 tcgcccacag tccccgagaa gttgggggga ggggtcggca attgatccgg tgcctagaga
     2461 aggtggcgcg gggtaaactg ggaaagtgat gtcgtgtact ggctccgcct ttttcccgag
     2521 ggtgggggag aaccgtatat aagtgcagta gtcgccgtga acgttctttt tcgcaacggg
     2581 tttgccgcca gaacacagga ccggttctag agcgctgcca ccatggacaa gaagtacagc
     2641 atcggcctgg acatcggcac caactctgtg ggctgggccg tgatcaccga cgagtacaag
     2701 gtgcccagca agaaattcaa ggtgctgggc aacaccgacc ggcacagcat caagaagaac
     2761 ctgatcggag ccctgctgtt cgacagcggc gaaacagccg aggccacccg gctgaagaga
     2821 accgccagaa gaagatacac cagacggaag aaccggatct gctatctgca agagatcttc
     2881 agcaacgaga tggccaaggt ggacgacagc ttcttccaca gactggaaga gtccttcctg
     2941 gtggaagagg ataagaagca cgagcggcac cccatcttcg gcaacatcgt ggacgaggtg
     3001 gcctaccacg agaagtaccc caccatctac cacctgagaa agaaactggt ggacagcacc
     3061 gacaaggccg acctgcggct gatctatctg gccctggccc acatgatcaa gttccggggc
     3121 cacttcctga tcgagggcga cctgaacccc gacaacagcg acgtggacaa gctgttcatc
     3181 cagctggtgc agacctacaa ccagctgttc gaggaaaacc ccatcaacgc cagcggcgtg
     3241 gacgccaagg ccatcctgtc tgccagactg agcaagagca gacggctgga aaatctgatc
     3301 gcccagctgc ccggcgagaa gaagaatggc ctgttcggaa acctgattgc cctgagcctg
     3361 ggcctgaccc ccaacttcaa gagcaacttc gacctggccg aggatgccaa actgcagctg
     3421 agcaaggaca cctacgacga cgacctggac aacctgctgg cccagatcgg cgaccagtac
     3481 gccgacctgt ttctggccgc caagaacctg tccgacgcca tcctgctgag cgacatcctg
     3541 agagtgaaca ccgagatcac caaggccccc ctgagcgcct ctatgatcaa gagatacgac
     3601 gagcaccacc aggacctgac cctgctgaaa gctctcgtgc ggcagcagct gcctgagaag
     3661 tacaaagaga ttttcttcga ccagagcaag aacggctacg ccggctacat tgacggcgga
     3721 gccagccagg aagagttcta caagttcatc aagcccatcc tggaaaagat ggacggcacc
     3781 gaggaactgc tcgtgaagct gaacagagag gacctgctgc ggaagcagcg gaccttcgac
     3841 aacggcagca tcccccacca gatccacctg ggagagctgc acgccattct gcggcggcag
     3901 gaagattttt acccattcct gaaggacaac cgggaaaaga tcgagaagat cctgaccttc
     3961 cgcatcccct actacgtggg ccctctggcc aggggaaaca gcagattcgc ctggatgacc
     4021 agaaagagcg aggaaaccat caccccctgg aacttcgagg aagtggtgga caagggcgct
     4081 tccgcccaga gcttcatcga gcggatgacc aacttcgata agaacctgcc caacgagaag
     4141 gtgctgccca agcacagcct gctgtacgag tacttcaccg tgtataacga gctgaccaaa
     4201 gtgaaatacg tgaccgaggg aatgagaaag cccgccttcc tgagcggcga gcagaaaaag
     4261 gccatcgtgg acctgctgtt caagaccaac cggaaagtga ccgtgaagca gctgaaagag
     4321 gactacttca agaaaatcga gtgcttcgac tccgtggaaa tctccggcgt ggaagatcgg
     4381 ttcaacgcct ccctgggcac ataccacgat ctgctgaaaa ttatcaagga caaggacttc
     4441 ctggacaatg aggaaaacga ggacattctg gaagatatcg tgctgaccct gacactgttt
     4501 gaggacagag agatgatcga ggaacggctg aaaacctatg cccacctgtt cgacgacaaa
     4561 gtgatgaagc agctgaagcg gcggagatac accggctggg gcaggctgag ccggaagctg
     4621 atcaacggca tccgggacaa gcagtccggc aagacaatcc tggatttcct gaagtccgac
     4681 ggcttcgcca acagaaactt catgcagctg atccacgacg acagcctgac ctttaaagag
     4741 gacatccaga aagcccaggt gtccggccag ggcgatagcc tgcacgagca cattgccaat
     4801 ctggccggca gccccgccat taagaagggc atcctgcaga cagtgaaggt ggtggacgag
     4861 ctcgtgaaag tgatgggccg gcacaagccc gagaacatcg tgatcgaaat ggccagagag
     4921 aaccagacca cccagaaggg acagaagaac agccgcgaga gaatgaagcg gatcgaagag
     4981 ggcatcaaag agctgggcag ccagatcctg aaagaacacc ccgtggaaaa cacccagctg
     5041 cagaacgaga agctgtacct gtactacctg cagaatgggc gggatatgta cgtggaccag
     5101 gaactggaca tcaaccggct gtccgactac gatgtggacc atatcgtgcc tcagagcttt
     5161 ctgaaggacg actccatcga caacaaggtg ctgaccagaa gcgacaagaa ccggggcaag
     5221 agcgacaacg tgccctccga agaggtcgtg aagaagatga agaactactg gcggcagctg
     5281 ctgaacgcca agctgattac ccagagaaag ttcgacaatc tgaccaaggc cgagagaggc
     5341 ggcctgagcg aactggataa ggccggcttc atcaagagac agctggtgga aacccggcag
     5401 atcacaaagc acgtggcaca gatcctggac tcccggatga acactaagta cgacgagaat
     5461 gacaagctga tccgggaagt gaaagtgatc accctgaagt ccaagctggt gtccgatttc
     5521 cggaaggatt tccagtttta caaagtgcgc gagatcaaca actaccacca cgcccacgac
     5581 gcctacctga acgccgtcgt gggaaccgcc ctgatcaaaa agtaccctaa gctggaaagc
     5641 gagttcgtgt acggcgacta caaggtgtac gacgtgcgga agatgatcgc caagagcgag
     5701 caggaaatcg gcaaggctac cgccaagtac ttcttctaca gcaacatcat gaactttttc
     5761 aagaccgaga ttaccctggc caacggcgag atccggaagc ggcctctgat cgagacaaac
     5821 ggcgaaaccg gggagatcgt gtgggataag ggccgggatt ttgccaccgt gcggaaagtg
     5881 ctgagcatgc cccaagtgaa tatcgtgaaa aagaccgagg tgcagacagg cggcttcagc
     5941 aaagagtcta tcctgcccaa gaggaacagc gataagctga tcgccagaaa gaaggactgg
     6001 gaccctaaga agtacggcgg cttcgacagc cccaccgtgg cctattctgt gctggtggtg
     6061 gccaaagtgg aaaagggcaa gtccaagaaa ctgaagagtg tgaaagagct gctggggatc
     6121 accatcatgg aaagaagcag cttcgagaag aatcccatcg actttctgga agccaagggc
     6181 tacaaagaag tgaaaaagga cctgatcatc aagctgccta agtactccct gttcgagctg
     6241 gaaaacggcc ggaagagaat gctggcctct gccggcgaac tgcagaaggg aaacgaactg
     6301 gccctgccct ccaaatatgt gaacttcctg tacctggcca gccactatga gaagctgaag
     6361 ggctcccccg aggataatga gcagaaacag ctgtttgtgg aacagcacaa gcactacctg
     6421 gacgagatca tcgagcagat cagcgagttc tccaagagag tgatcctggc cgacgctaat
     6481 ctggacaaag tgctgtccgc ctacaacaag caccgggata agcccatcag agagcaggcc
     6541 gagaatatca tccacctgtt taccctgacc aatctgggag cccctgccgc cttcaagtac
     6601 tttgacacca ccatcgaccg gaagaggtac accagcacca aagaggtgct ggacgccacc
     6661 ctgatccacc agagcatcac cggcctgtac gagacacgga tcgacctgtc tcagctggga
     6721 ggcgacaagc gacctgccgc cacaaagaag gctggacagg ctaagaagaa gaaagattac
     6781 aaagacgatg acgataaggg atccggcgca acaaacttct ctctgctgaa acaagccgga
     6841 gatgtcgaag agaatcctgg accgaccgag tacaagccca cggtgcgcct cgccacccgc
     6901 gacgacgtcc ccagggccgt acgcaccctc gccgccgcgt tcgccgacta ccccgccacg
     6961 cgccacaccg tcgatccgga ccgccacatc gagcgggtca ccgagctgca agaactcttc
     7021 ctcacgcgcg tcgggctcga catcggcaag gtgtgggtcg cggacgacgg cgccgcggtg
     7081 gcggtctgga ccacgccgga gagcgtcgaa gcgggggcgg tgttcgccga gatcggcccg
     7141 cgcatggccg agttgagcgg ttcccggctg gccgcgcagc aacagatgga aggcctcctg
     7201 gcgccgcacc ggcccaagga gcccgcgtgg ttcctggcca ccgtcggagt ctcgcccgac
     7261 caccagggca agggtctggg cagcgccgtc gtgctccccg gagtggaggc ggccgagcgc
     7321 gccggggtgc ccgccttcct ggagacctcc gcgccccgca acctcccctt ctacgagcgg
     7381 ctcggcttca ccgtcaccgc cgacgtcgag gtgcccgaag gaccgcgcac ctggtgcatg
     7441 acccgcaagc ccggtgcctg aacgcgttaa gtcgacaatc aacctctgga ttacaaaatt
     7501 tgtgaaagat tgactggtat tcttaactat gttgctcctt ttacgctatg tggatacgct
     7561 gctttaatgc ctttgtatca tgctattgct tcccgtatgg ctttcatttt ctcctccttg
     7621 tataaatcct ggttgctgtc tctttatgag gagttgtggc ccgttgtcag gcaacgtggc
     7681 gtggtgtgca ctgtgtttgc tgacgcaacc cccactggtt ggggcattgc caccacctgt
     7741 cagctccttt ccgggacttt cgctttcccc ctccctattg ccacggcgga actcatcgcc
     7801 gcctgccttg cccgctgctg gacaggggct cggctgttgg gcactgacaa ttccgtggtg
     7861 ttgtcgggga aatcatcgtc ctttccttgg ctgctcgcct gtgttgccac ctggattctg
     7921 cgcgggacgt ccttctgcta cgtcccttcg gccctcaatc cagcggacct tccttcccgc
     7981 ggcctgctgc cggctctgcg gcctcttccg cgtcttcgcc ttcgccctca gacgagtcgg
     8041 atctcccttt gggccgcctc cccgcgtcga ctttaagacc aatgacttac aaggcagctg
     8101 tagatcttag ccacttttta aaagaaaagg ggggactgga agggctaatt cactcccaac
     8161 gaagacaaga tctgcttttt gcttgtactg ggtctctctg gttagaccag atctgagcct
     8221 gggagctctc tggctaacta gggaacccac tgcttaagcc tcaataaagc ttgccttgag
     8281 tgcttcaagt agtgtgtgcc cgtctgttgt gtgactctgg taactagaga tccctcagac
     8341 ccttttagtc agtgtggaaa atctctagca gggcccgttt aaacccgctg atcagcctcg
     8401 actgtgcctt ctagttgcca gccatctgtt gtttgcccct cccccgtgcc ttccttgacc
     8461 ctggaaggtg ccactcccac tgtcctttcc taataaaatg aggaaattgc atcgcattgt
     8521 ctgagtaggt gtcattctat tctggggggt ggggtggggc aggacagcaa gggggaggat
     8581 tgggaagaca atagcaggca tgctggggat gcggtgggct ctatggcttc tgaggcggaa
     8641 agaaccagct ggggctctag ggggtatccc cacgcgccct gtagcggcgc attaagcgcg
     8701 gcgggtgtgg tggttacgcg cagcgtgacc gctacacttg ccagcgccct agcgcccgct
     8761 cctttcgctt tcttcccttc ctttctcgcc acgttcgccg gctttccccg tcaagctcta
     8821 aatcgggggc tccctttagg gttccgattt agtgctttac ggcacctcga ccccaaaaaa
     8881 cttgattagg gtgatggttc acgtagtggg ccatcgccct gatagacggt ttttcgccct
     8941 ttgacgttgg agtccacgtt ctttaatagt ggactcttgt tccaaactgg aacaacactc
     9001 aaccctatct cggtctattc ttttgattta taagggattt tgccgatttc ggcctattgg
     9061 ttaaaaaatg agctgattta acaaaaattt aacgcgaatt aattctgtgg aatgtgtgtc
     9121 agttagggtg tggaaagtcc ccaggctccc cagcaggcag aagtatgcaa agcatgcatc
     9181 tcaattagtc agcaaccagg tgtggaaagt ccccaggctc cccagcaggc agaagtatgc
     9241 aaagcatgca tctcaattag tcagcaacca tagtcccgcc cctaactccg cccatcccgc
     9301 ccctaactcc gcccagttcc gcccattctc cgccccatgg ctgactaatt ttttttattt
     9361 atgcagaggc cgaggccgcc tctgcctctg agctattcca gaagtagtga ggaggctttt
     9421 ttggaggcct aggcttttgc aaaaagctcc cgggagcttg tatatccatt ttcggatctg
     9481 atcagcacgt gttgacaatt aatcatcggc atagtatatc ggcatagtat aatacgacaa
     9541 ggtgaggaac taaaccatgg ccaagttgac cagtgccgtt ccggtgctca ccgcgcgcga
     9601 cgtcgccgga gcggtcgagt tctggaccga ccggctcggg ttctcccggg acttcgtgga
     9661 ggacgacttc gccggtgtgg tccgggacga cgtgaccctg ttcatcagcg cggtccagga
     9721 ccaggtggtg ccggacaaca ccctggcctg ggtgtgggtg cgcggcctgg acgagctgta
     9781 cgccgagtgg tcggaggtcg tgtccacgaa cttccgggac gcctccgggc cggccatgac
     9841 cgagatcggc gagcagccgt gggggcggga gttcgccctg cgcgacccgg ccggcaactg
     9901 cgtgcacttc gtggccgagg agcaggactg acacgtgcta cgagatttcg attccaccgc
     9961 cgccttctat gaaaggttgg gcttcggaat cgttttccgg gacgccggct ggatgatcct
    10021 ccagcgcggg gatctcatgc tggagttctt cgcccacccc aacttgttta ttgcagctta
    10081 taatggttac aaataaagca atagcatcac aaatttcaca aataaagcat ttttttcact
    10141 gcattctagt tgtggtttgt ccaaactcat caatgtatct tatcatgtct gtataccgtc
    10201 gacctctagc tagagcttgg cgtaatcatg gtcatagctg tttcctgtgt gaaattgtta
    10261 tccgctcaca attccacaca acatacgagc cggaagcata aagtgtaaag cctggggtgc
    10321 ctaatgagtg agctaactca cattaattgc gttgcgctca ctgcccgctt tccagtcggg
    10381 aaacctgtcg tgccagctgc attaatgaat cggccaacgc gcggggagag gcggtttgcg
    10441 tattgggcgc tcttccgctt cctcgctcac tgactcgctg cgctcggtcg ttcggctgcg
    10501 gcgagcggta tcagctcact caaaggcggt aatacggtta tccacagaat caggggataa
    10561 cgcaggaaag aacatgtgag caaaaggcca gcaaaaggcc aggaaccgta aaaaggccgc
    10621 gttgctggcg tttttccata ggctccgccc ccctgacgag catcacaaaa atcgacgctc
    10681 aagtcagagg tggcgaaacc cgacaggact ataaagatac caggcgtttc cccctggaag
    10741 ctccctcgtg cgctctcctg ttccgaccct gccgcttacc ggatacctgt ccgcctttct
    10801 cccttcggga agcgtggcgc tttctcatag ctcacgctgt aggtatctca gttcggtgta
    10861 ggtcgttcgc tccaagctgg gctgtgtgca cgaacccccc gttcagcccg accgctgcgc
    10921 cttatccggt aactatcgtc ttgagtccaa cccggtaaga cacgacttat cgccactggc
    10981 agcagccact ggtaacagga ttagcagagc gaggtatgta ggcggtgcta cagagttctt
    11041 gaagtggtgg cctaactacg gctacactag aagaacagta tttggtatct gcgctctgct
    11101 gaagccagtt accttcggaa aaagagttgg tagctcttga tccggcaaac aaaccaccgc
    11161 tggtagcggt ggtttttttg tttgcaagca gcagattacg cgcagaaaaa aaggatctca
    11221 agaagatcct ttgatctttt ctacggggtc tgacgctcag tggaacgaaa actcacgtta
    11281 agggattttg gtcatgagat tatcaaaaag gatcttcacc tagatccttt taaattaaaa
    11341 atgaagtttt aaatcaatct aaagtatata tgagtaaact tggtctgaca gttaccaatg
    11401 cttaatcagt gaggcaccta tctcagcgat ctgtctattt cgttcatcca tagttgcctg
    11461 actccccgtc gtgtagataa ctacgatacg ggagggctta ccatctggcc ccagtgctgc
    11521 aatgataccg cgagacccac gctcaccggc tccagattta tcagcaataa accagccagc
    11581 cggaagggcc gagcgcagaa gtggtcctgc aactttatcc gcctccatcc agtctattaa
    11641 ttgttgccgg gaagctagag taagtagttc gccagttaat agtttgcgca acgttgttgc
    11701 cattgctaca ggcatcgtgg tgtcacgctc gtcgtttggt atggcttcat tcagctccgg
    11761 ttcccaacga tcaaggcgag ttacatgatc ccccatgttg tgcaaaaaag cggttagctc
    11821 cttcggtcct ccgatcgttg tcagaagtaa gttggccgca gtgttatcac tcatggttat
    11881 ggcagcactg cataattctc ttactgtcat gccatccgta agatgctttt ctgtgactgg
    11941 tgagtactca accaagtcat tctgagaata gtgtatgcgg cgaccgagtt gctcttgccc
    12001 ggcgtcaata cgggataata ccgcgccaca tagcagaact ttaaaagtgc tcatcattgg
    12061 aaaacgttct tcggggcgaa aactctcaag gatcttaccg ctgttgagat ccagttcgat
    12121 gtaacccact cgtgcaccca actgatcttc agcatctttt actttcacca gcgtttctgg
    12181 gtgagcaaaa acaggaaggc aaaatgccgc aaaaaaggga ataagggcga cacggaaatg
    12241 ttgaatactc atactcttcc tttttcaata ttattgaagc atttatcagg gttattgtct
    12301 catgagcgga tacatatttg aatgtattta gaaaaataaa caaatagggg ttccgcgcac
    12361 atttccccga aaagtgccac ctgacgtcga cggatcggga gatctcccga tcccctatgg
    12421 tgcactctca gtacaatctg ctctgatgcc gcatagttaa gccagtatct gctccctgct
    12481 tgtgtgttgg aggtcgctga gtagtgcgcg agcaaaattt aagctacaac aaggcaaggc
    12541 ttgaccgaca attgcatgaa gaatctgctt agggttaggc gttttgcgct gcttcgcgat
    12601 gtacgggcca gatatacgcg ttgacattga ttattgacta gttattaata gtaatcaatt
    12661 acggggtcat tagttcatag cccatatatg gagttccgcg ttacataact tacggtaaat
    12721 ggcccgcctg gctgaccgcc caacgacccc cgcccattga cgtcaataat gacgtatgtt
    12781 cccatagtaa cgccaatagg gactttccat tgacgtcaat gggtggagta tttacggtaa
    12841 actgcccact tggcagtaca tcaagtgtat catatgccaa gtacgccccc tattgacgtc
    12901 aatgacggta aatggcccgc ctggcattat gcccagtaca tgaccttatg ggactttcct
    12961 acttggcagt acatctacgt attagtcatc gctattacca tggtgatgcg gtt
//
