LOCUS       sequence-413626-       13013 bp    DNA     circular SYN 15-DEC-2025
DEFINITION  knock out HSPC300 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-sgHSPC300-2
FEATURES             Location/Qualifiers
     source          1..13013
                     /mol_type="other DNA"
                     /organism="synthetic DNA construct"
     promoter        21..219
                     /label=CMV promoter
                     /note="human cytomegalovirus (CMV) immediate early 
                     promoter"
     primer_bind     174..194
                     /label=CMV-F
                     /note="Human CMV immediate early promoter, forward primer"
     LTR             237..417
                     /label=5' LTR (truncated)
                     /note="truncated 5' long terminal repeat (LTR) from HIV-1"
     misc_feature    464..589
                     /label=HIV-1 Psi
                     /note="packaging signal of human immunodeficiency virus 
                     type 1"
     misc_feature    1082..1315
                     /label=RRE
                     /note="The Rev response element (RRE) of HIV-1 allows for 
                     Rev-dependent mRNA export from the nucleus to the 
                     cytoplasm."
     CDS             1500..1544
                     /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    1842..1959
                     /label=cPPT/CTS
                     /note="central polypurine tract and central termination 
                     sequence of HIV-1"
     promoter        2010..2258
                     /label=U6 promoter
                     /note="RNA polymerase III promoter for human U6 snRNA 
                     (Domitrovich & Kunkel, 2003)"
     primer_bind     2010..2030
                     /label=hU6-F
                     /note="Human U6 promoter, forward primer"
     primer_bind     2181..2200
                     /label=LKO.1 5'
                     /note="Human U6 promoter, forward primer"
     misc_RNA        2280..2355
                     /label=gRNA scaffold
                     /note="guide RNA scaffold for the Streptococcus pyogenes 
                     CRISPR/Cas9 system"
     promoter        2417..2628
                     /label=EF-1-alpha core promoter
                     /note="core promoter for human elongation factor 
                     EF-1-alpha"
     regulatory      2647..2656
                     /label=Kozak sequence
                     /note="vertebrate consensus sequence for strong initiation 
                     of translation (Kozak, 1987)"
                     /regulatory_class="other"
     CDS             2653..6756
                     /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             2653..6756
                     /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             6757..6804
                     /codon_start=1
                     /product="bipartite nuclear localization signal from 
                     nucleoplasmin"
                     /label=nucleoplasmin NLS
                     /translation="KRPAATKKAGQAKKKK"
     CDS             6805..6828
                     /codon_start=1
                     /product="FLAG(R) epitope tag, followed by an enterokinase 
                     cleavage site"
                     /label=FLAG
                     /translation="DYKDDDDK"
     CDS             6838..6894
                     /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             6895..7491
                     /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     7388..7408
                     /label=Puro-F
                     /note="Puromycin resistance gene, forward primer"
     misc_feature    7507..8095
                     /label=WPRE
                     /note="woodchuck hepatitis virus posttranscriptional 
                     regulatory element"
     primer_bind     complement(7560..7580)
                     /label=WPRE-R
                     /note="WPRE, reverse primer"
     CDS             complement(7978..7989)
                     /codon_start=1
                     /product="Factor Xa recognition and cleavage site"
                     /label=Factor Xa site
                     /translation="IEGR"
     LTR             8167..8400
                     /label=3' LTR (Delta-U3)
                     /note="self-inactivating 3' long terminal repeat (LTR) from
                     HIV-1"
     primer_bind     complement(8426..8443)
                     /label=BGH-rev
                     /note="Bovine growth hormone terminator, reverse primer. 
                     Also called BGH reverse"
     polyA_signal    8432..8656
                     /label=bGH poly(A) signal
                     /note="bovine growth hormone polyadenylation signal"
     rep_origin      8702..9130
                     /direction=RIGHT
                     /label=f1 ori
                     /note="f1 bacteriophage origin of replication; arrow 
                     indicates direction of (+) strand synthesis"
     primer_bind     complement(8789..8808)
                     /label=F1ori-R
                     /note="F1 origin, reverse primer"
     primer_bind     8999..9020
                     /label=F1ori-F
                     /note="F1 origin, forward primer"
     primer_bind     complement(9139..9159)
                     /label=pBABE 3'
                     /note="SV40 enhancer, reverse primer for pBABE vectors"
     promoter        9144..9473
                     /label=SV40 promoter
                     /note="SV40 enhancer and early promoter"
     rep_origin      9324..9459
                     /label=SV40 ori
                     /note="SV40 origin of replication"
     primer_bind     9386..9405
                     /label=SV40pro-F
                     /note="SV40 promoter/origin, forward primer"
     promoter        9521..9568
                     /label=EM7 promoter
                     /note="synthetic bacterial promoter "
     CDS             9587..9961
                     /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    10091..10212
                     /label=SV40 poly(A) signal
                     /note="SV40 polyadenylation signal"
     primer_bind     complement(10128..10147)
                     /label=SV40pA-R
                     /note="SV40 polyA, reverse primer"
     primer_bind     10182..10201
                     /label=EBV-rev
                     /note="SV40 polyA terminator, reverse primer"
     primer_bind     complement(10261..10277)
                     /label=M13 rev
                     /note="common sequencing primer, one of multiple similar 
                     variants"
     primer_bind     complement(10261..10277)
                     /label=M13 Reverse
                     /note="In lacZ gene. Also called M13-rev"
     primer_bind     complement(10274..10296)
                     /label=M13/pUC Reverse
                     /note="In lacZ gene"
     protein_bind    10285..10301
                     /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(10309..10315,10316..10333,10334..10339))
                     /label=lac promoter
                     /note="promoter for the E. coli lac operon"
     protein_bind    10354..10375
                     /label=CAP binding site
                     /bound_moiety="E. coli catabolite activator protein"
                     /note="CAP binding activates transcription in the presence 
                     of cAMP."
     primer_bind     complement(10492..10509)
                     /label=L4440
                     /note="L4440 vector, forward primer"
     rep_origin      complement(10663..11251)
                     /direction=LEFT
                     /label=ori
                     /note="high-copy-number ColE1/pMB1/pBR322/pUC origin of 
                     replication"
     primer_bind     complement(10743..10762)
                     /label=pBR322ori-F
                     /note="pBR322 origin, forward primer"
     CDS             complement(join(11422..12213,12214..12282))
                     /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     12045..12064
                     /label=Amp-R
                     /note="Ampicillin resistance gene, reverse primer"
     promoter        complement(12283..12387)
                     /gene="bla"
                     /label=AmpR promoter
     primer_bind     complement(12462..12481)
                     /label=pRS-marker
                     /note="pRS vectors, use to sequence yeast selectable 
                     marker"
     enhancer        join(12653..13013,1..19)
                     /label=CMV enhancer
                     /note="human cytomegalovirus immediate early enhancer"
ORIGIN
        1 agtcatcgct attaccatgg tgatgcggtt ttggcagtac atcaatgggc gtggatagcg
       61 gtttgactca cggggatttc caagtctcca ccccattgac gtcaatggga gtttgttttg
      121 gcaccaaaat caacgggact ttccaaaatg tcgtaacaac tccgccccat tgacgcaaat
      181 gggcggtagg cgtgtacggt gggaggtcta tataagcagc gcgttttgcc tgtactgggt
      241 ctctctggtt agaccagatc tgagcctggg agctctctgg ctaactaggg aacccactgc
      301 ttaagcctca ataaagcttg ccttgagtgc ttcaagtagt gtgtgcccgt ctgttgtgtg
      361 actctggtaa ctagagatcc ctcagaccct tttagtcagt gtggaaaatc tctagcagtg
      421 gcgcccgaac agggacttga aagcgaaagg gaaaccagag gagctctctc gacgcaggac
      481 tcggcttgct gaagcgcgca cggcaagagg cgaggggcgg cgactggtga gtacgccaaa
      541 aattttgact agcggaggct agaaggagag agatgggtgc gagagcgtca gtattaagcg
      601 ggggagaatt agatcgcgat gggaaaaaat tcggttaagg ccagggggaa agaaaaaata
      661 taaattaaaa catatagtat gggcaagcag ggagctagaa cgattcgcag ttaatcctgg
      721 cctgttagaa acatcagaag gctgtagaca aatactggga cagctacaac catcccttca
      781 gacaggatca gaagaactta gatcattata taatacagta gcaaccctct attgtgtgca
      841 tcaaaggata gagataaaag acaccaagga agctttagac aagatagagg aagagcaaaa
      901 caaaagtaag accaccgcac agcaagcggc cgctgatctt cagacctgga ggaggagata
      961 tgagggacaa ttggagaagt gaattatata aatataaagt agtaaaaatt gaaccattag
     1021 gagtagcacc caccaaggca aagagaagag tggtgcagag agaaaaaaga gcagtgggaa
     1081 taggagcttt gttccttggg ttcttgggag cagcaggaag cactatgggc gcagcgtcaa
     1141 tgacgctgac ggtacaggcc agacaattat tgtctggtat agtgcagcag cagaacaatt
     1201 tgctgagggc tattgaggcg caacagcatc tgttgcaact cacagtctgg ggcatcaagc
     1261 agctccaggc aagaatcctg gctgtggaaa gatacctaaa ggatcaacag ctcctgggga
     1321 tttggggttg ctctggaaaa ctcatttgca ccactgctgt gccttggaat gctagttgga
     1381 gtaataaatc tctggaacag atttggaatc acacgacctg gatggagtgg gacagagaaa
     1441 ttaacaatta cacaagctta atacactcct taattgaaga atcgcaaaac cagcaagaaa
     1501 agaatgaaca agaattattg gaattagata aatgggcaag tttgtggaat tggtttaaca
     1561 taacaaattg gctgtggtat ataaaattat tcataatgat agtaggaggc ttggtaggtt
     1621 taagaatagt ttttgctgta ctttctatag tgaatagagt taggcaggga tattcaccat
     1681 tatcgtttca gacccacctc ccaaccccga ggggacccga caggcccgaa ggaatagaag
     1741 aagaaggtgg agagagagac agagacagat ccattcgatt agtgaacgga tcggcactgc
     1801 gtgcgccaat tctgcagaca aatggcagta ttcatccaca attttaaaag aaaagggggg
     1861 attggggggt acagtgcagg ggaaagaata gtagacataa tagcaacaga catacaaact
     1921 aaagaattac aaaaacaaat tacaaaaatt caaaattttc gggtttatta cagggacagc
     1981 agagatccag tttggttaat taaggtaccg agggcctatt tcccatgatt ccttcatatt
     2041 tgcatatacg atacaaggct gttagagaga taattagaat taatttgact gtaaacacaa
     2101 agatattagt acaaaatacg tgacgtagaa agtaataatt tcttgggtag tttgcagttt
     2161 taaaattatg ttttaaaatg gactatcata tgcttaccgt aacttgaaag tatttcgatt
     2221 tcttggcttt atatatcttg tggaaaggac gaaacaccgt gattatctca atgtactccg
     2281 ttttagagct agaaatagca agttaaaata aggctagtcc gttatcaact tgaaaaagtg
     2341 gcaccgagtc ggtgcttttt tgaattcgct agctaggtct tgaaaggagt gggaattggc
     2401 tccggtgccc gtcagtgggc agagcgcaca tcgcccacag tccccgagaa gttgggggga
     2461 ggggtcggca attgatccgg tgcctagaga aggtggcgcg gggtaaactg ggaaagtgat
     2521 gtcgtgtact ggctccgcct ttttcccgag ggtgggggag aaccgtatat aagtgcagta
     2581 gtcgccgtga acgttctttt tcgcaacggg tttgccgcca gaacacagga ccggttctag
     2641 agcgctgcca ccatggacaa gaagtacagc atcggcctgg acatcggcac caactctgtg
     2701 ggctgggccg tgatcaccga cgagtacaag gtgcccagca agaaattcaa ggtgctgggc
     2761 aacaccgacc ggcacagcat caagaagaac ctgatcggag ccctgctgtt cgacagcggc
     2821 gaaacagccg aggccacccg gctgaagaga accgccagaa gaagatacac cagacggaag
     2881 aaccggatct gctatctgca agagatcttc agcaacgaga tggccaaggt ggacgacagc
     2941 ttcttccaca gactggaaga gtccttcctg gtggaagagg ataagaagca cgagcggcac
     3001 cccatcttcg gcaacatcgt ggacgaggtg gcctaccacg agaagtaccc caccatctac
     3061 cacctgagaa agaaactggt ggacagcacc gacaaggccg acctgcggct gatctatctg
     3121 gccctggccc acatgatcaa gttccggggc cacttcctga tcgagggcga cctgaacccc
     3181 gacaacagcg acgtggacaa gctgttcatc cagctggtgc agacctacaa ccagctgttc
     3241 gaggaaaacc ccatcaacgc cagcggcgtg gacgccaagg ccatcctgtc tgccagactg
     3301 agcaagagca gacggctgga aaatctgatc gcccagctgc ccggcgagaa gaagaatggc
     3361 ctgttcggaa acctgattgc cctgagcctg ggcctgaccc ccaacttcaa gagcaacttc
     3421 gacctggccg aggatgccaa actgcagctg agcaaggaca cctacgacga cgacctggac
     3481 aacctgctgg cccagatcgg cgaccagtac gccgacctgt ttctggccgc caagaacctg
     3541 tccgacgcca tcctgctgag cgacatcctg agagtgaaca ccgagatcac caaggccccc
     3601 ctgagcgcct ctatgatcaa gagatacgac gagcaccacc aggacctgac cctgctgaaa
     3661 gctctcgtgc ggcagcagct gcctgagaag tacaaagaga ttttcttcga ccagagcaag
     3721 aacggctacg ccggctacat tgacggcgga gccagccagg aagagttcta caagttcatc
     3781 aagcccatcc tggaaaagat ggacggcacc gaggaactgc tcgtgaagct gaacagagag
     3841 gacctgctgc ggaagcagcg gaccttcgac aacggcagca tcccccacca gatccacctg
     3901 ggagagctgc acgccattct gcggcggcag gaagattttt acccattcct gaaggacaac
     3961 cgggaaaaga tcgagaagat cctgaccttc cgcatcccct actacgtggg ccctctggcc
     4021 aggggaaaca gcagattcgc ctggatgacc agaaagagcg aggaaaccat caccccctgg
     4081 aacttcgagg aagtggtgga caagggcgct tccgcccaga gcttcatcga gcggatgacc
     4141 aacttcgata agaacctgcc caacgagaag gtgctgccca agcacagcct gctgtacgag
     4201 tacttcaccg tgtataacga gctgaccaaa gtgaaatacg tgaccgaggg aatgagaaag
     4261 cccgccttcc tgagcggcga gcagaaaaag gccatcgtgg acctgctgtt caagaccaac
     4321 cggaaagtga ccgtgaagca gctgaaagag gactacttca agaaaatcga gtgcttcgac
     4381 tccgtggaaa tctccggcgt ggaagatcgg ttcaacgcct ccctgggcac ataccacgat
     4441 ctgctgaaaa ttatcaagga caaggacttc ctggacaatg aggaaaacga ggacattctg
     4501 gaagatatcg tgctgaccct gacactgttt gaggacagag agatgatcga ggaacggctg
     4561 aaaacctatg cccacctgtt cgacgacaaa gtgatgaagc agctgaagcg gcggagatac
     4621 accggctggg gcaggctgag ccggaagctg atcaacggca tccgggacaa gcagtccggc
     4681 aagacaatcc tggatttcct gaagtccgac ggcttcgcca acagaaactt catgcagctg
     4741 atccacgacg acagcctgac ctttaaagag gacatccaga aagcccaggt gtccggccag
     4801 ggcgatagcc tgcacgagca cattgccaat ctggccggca gccccgccat taagaagggc
     4861 atcctgcaga cagtgaaggt ggtggacgag ctcgtgaaag tgatgggccg gcacaagccc
     4921 gagaacatcg tgatcgaaat ggccagagag aaccagacca cccagaaggg acagaagaac
     4981 agccgcgaga gaatgaagcg gatcgaagag ggcatcaaag agctgggcag ccagatcctg
     5041 aaagaacacc ccgtggaaaa cacccagctg cagaacgaga agctgtacct gtactacctg
     5101 cagaatgggc gggatatgta cgtggaccag gaactggaca tcaaccggct gtccgactac
     5161 gatgtggacc atatcgtgcc tcagagcttt ctgaaggacg actccatcga caacaaggtg
     5221 ctgaccagaa gcgacaagaa ccggggcaag agcgacaacg tgccctccga agaggtcgtg
     5281 aagaagatga agaactactg gcggcagctg ctgaacgcca agctgattac ccagagaaag
     5341 ttcgacaatc tgaccaaggc cgagagaggc ggcctgagcg aactggataa ggccggcttc
     5401 atcaagagac agctggtgga aacccggcag atcacaaagc acgtggcaca gatcctggac
     5461 tcccggatga acactaagta cgacgagaat gacaagctga tccgggaagt gaaagtgatc
     5521 accctgaagt ccaagctggt gtccgatttc cggaaggatt tccagtttta caaagtgcgc
     5581 gagatcaaca actaccacca cgcccacgac gcctacctga acgccgtcgt gggaaccgcc
     5641 ctgatcaaaa agtaccctaa gctggaaagc gagttcgtgt acggcgacta caaggtgtac
     5701 gacgtgcgga agatgatcgc caagagcgag caggaaatcg gcaaggctac cgccaagtac
     5761 ttcttctaca gcaacatcat gaactttttc aagaccgaga ttaccctggc caacggcgag
     5821 atccggaagc ggcctctgat cgagacaaac ggcgaaaccg gggagatcgt gtgggataag
     5881 ggccgggatt ttgccaccgt gcggaaagtg ctgagcatgc cccaagtgaa tatcgtgaaa
     5941 aagaccgagg tgcagacagg cggcttcagc aaagagtcta tcctgcccaa gaggaacagc
     6001 gataagctga tcgccagaaa gaaggactgg gaccctaaga agtacggcgg cttcgacagc
     6061 cccaccgtgg cctattctgt gctggtggtg gccaaagtgg aaaagggcaa gtccaagaaa
     6121 ctgaagagtg tgaaagagct gctggggatc accatcatgg aaagaagcag cttcgagaag
     6181 aatcccatcg actttctgga agccaagggc tacaaagaag tgaaaaagga cctgatcatc
     6241 aagctgccta agtactccct gttcgagctg gaaaacggcc ggaagagaat gctggcctct
     6301 gccggcgaac tgcagaaggg aaacgaactg gccctgccct ccaaatatgt gaacttcctg
     6361 tacctggcca gccactatga gaagctgaag ggctcccccg aggataatga gcagaaacag
     6421 ctgtttgtgg aacagcacaa gcactacctg gacgagatca tcgagcagat cagcgagttc
     6481 tccaagagag tgatcctggc cgacgctaat ctggacaaag tgctgtccgc ctacaacaag
     6541 caccgggata agcccatcag agagcaggcc gagaatatca tccacctgtt taccctgacc
     6601 aatctgggag cccctgccgc cttcaagtac tttgacacca ccatcgaccg gaagaggtac
     6661 accagcacca aagaggtgct ggacgccacc ctgatccacc agagcatcac cggcctgtac
     6721 gagacacgga tcgacctgtc tcagctggga ggcgacaagc gacctgccgc cacaaagaag
     6781 gctggacagg ctaagaagaa gaaagattac aaagacgatg acgataaggg atccggcgca
     6841 acaaacttct ctctgctgaa acaagccgga gatgtcgaag agaatcctgg accgaccgag
     6901 tacaagccca cggtgcgcct cgccacccgc gacgacgtcc ccagggccgt acgcaccctc
     6961 gccgccgcgt tcgccgacta ccccgccacg cgccacaccg tcgatccgga ccgccacatc
     7021 gagcgggtca ccgagctgca agaactcttc ctcacgcgcg tcgggctcga catcggcaag
     7081 gtgtgggtcg cggacgacgg cgccgcggtg gcggtctgga ccacgccgga gagcgtcgaa
     7141 gcgggggcgg tgttcgccga gatcggcccg cgcatggccg agttgagcgg ttcccggctg
     7201 gccgcgcagc aacagatgga aggcctcctg gcgccgcacc ggcccaagga gcccgcgtgg
     7261 ttcctggcca ccgtcggagt ctcgcccgac caccagggca agggtctggg cagcgccgtc
     7321 gtgctccccg gagtggaggc ggccgagcgc gccggggtgc ccgccttcct ggagacctcc
     7381 gcgccccgca acctcccctt ctacgagcgg ctcggcttca ccgtcaccgc cgacgtcgag
     7441 gtgcccgaag gaccgcgcac ctggtgcatg acccgcaagc ccggtgcctg aacgcgttaa
     7501 gtcgacaatc aacctctgga ttacaaaatt tgtgaaagat tgactggtat tcttaactat
     7561 gttgctcctt ttacgctatg tggatacgct gctttaatgc ctttgtatca tgctattgct
     7621 tcccgtatgg ctttcatttt ctcctccttg tataaatcct ggttgctgtc tctttatgag
     7681 gagttgtggc ccgttgtcag gcaacgtggc gtggtgtgca ctgtgtttgc tgacgcaacc
     7741 cccactggtt ggggcattgc caccacctgt cagctccttt ccgggacttt cgctttcccc
     7801 ctccctattg ccacggcgga actcatcgcc gcctgccttg cccgctgctg gacaggggct
     7861 cggctgttgg gcactgacaa ttccgtggtg ttgtcgggga aatcatcgtc ctttccttgg
     7921 ctgctcgcct gtgttgccac ctggattctg cgcgggacgt ccttctgcta cgtcccttcg
     7981 gccctcaatc cagcggacct tccttcccgc ggcctgctgc cggctctgcg gcctcttccg
     8041 cgtcttcgcc ttcgccctca gacgagtcgg atctcccttt gggccgcctc cccgcgtcga
     8101 ctttaagacc aatgacttac aaggcagctg tagatcttag ccacttttta aaagaaaagg
     8161 ggggactgga agggctaatt cactcccaac gaagacaaga tctgcttttt gcttgtactg
     8221 ggtctctctg gttagaccag atctgagcct gggagctctc tggctaacta gggaacccac
     8281 tgcttaagcc tcaataaagc ttgccttgag tgcttcaagt agtgtgtgcc cgtctgttgt
     8341 gtgactctgg taactagaga tccctcagac ccttttagtc agtgtggaaa atctctagca
     8401 gggcccgttt aaacccgctg atcagcctcg actgtgcctt ctagttgcca gccatctgtt
     8461 gtttgcccct cccccgtgcc ttccttgacc ctggaaggtg ccactcccac tgtcctttcc
     8521 taataaaatg aggaaattgc atcgcattgt ctgagtaggt gtcattctat tctggggggt
     8581 ggggtggggc aggacagcaa gggggaggat tgggaagaca atagcaggca tgctggggat
     8641 gcggtgggct ctatggcttc tgaggcggaa agaaccagct ggggctctag ggggtatccc
     8701 cacgcgccct gtagcggcgc attaagcgcg gcgggtgtgg tggttacgcg cagcgtgacc
     8761 gctacacttg ccagcgccct agcgcccgct cctttcgctt tcttcccttc ctttctcgcc
     8821 acgttcgccg gctttccccg tcaagctcta aatcgggggc tccctttagg gttccgattt
     8881 agtgctttac ggcacctcga ccccaaaaaa cttgattagg gtgatggttc acgtagtggg
     8941 ccatcgccct gatagacggt ttttcgccct ttgacgttgg agtccacgtt ctttaatagt
     9001 ggactcttgt tccaaactgg aacaacactc aaccctatct cggtctattc ttttgattta
     9061 taagggattt tgccgatttc ggcctattgg ttaaaaaatg agctgattta acaaaaattt
     9121 aacgcgaatt aattctgtgg aatgtgtgtc agttagggtg tggaaagtcc ccaggctccc
     9181 cagcaggcag aagtatgcaa agcatgcatc tcaattagtc agcaaccagg tgtggaaagt
     9241 ccccaggctc cccagcaggc agaagtatgc aaagcatgca tctcaattag tcagcaacca
     9301 tagtcccgcc cctaactccg cccatcccgc ccctaactcc gcccagttcc gcccattctc
     9361 cgccccatgg ctgactaatt ttttttattt atgcagaggc cgaggccgcc tctgcctctg
     9421 agctattcca gaagtagtga ggaggctttt ttggaggcct aggcttttgc aaaaagctcc
     9481 cgggagcttg tatatccatt ttcggatctg atcagcacgt gttgacaatt aatcatcggc
     9541 atagtatatc ggcatagtat aatacgacaa ggtgaggaac taaaccatgg ccaagttgac
     9601 cagtgccgtt ccggtgctca ccgcgcgcga cgtcgccgga gcggtcgagt tctggaccga
     9661 ccggctcggg ttctcccggg acttcgtgga ggacgacttc gccggtgtgg tccgggacga
     9721 cgtgaccctg ttcatcagcg cggtccagga ccaggtggtg ccggacaaca ccctggcctg
     9781 ggtgtgggtg cgcggcctgg acgagctgta cgccgagtgg tcggaggtcg tgtccacgaa
     9841 cttccgggac gcctccgggc cggccatgac cgagatcggc gagcagccgt gggggcggga
     9901 gttcgccctg cgcgacccgg ccggcaactg cgtgcacttc gtggccgagg agcaggactg
     9961 acacgtgcta cgagatttcg attccaccgc cgccttctat gaaaggttgg gcttcggaat
    10021 cgttttccgg gacgccggct ggatgatcct ccagcgcggg gatctcatgc tggagttctt
    10081 cgcccacccc aacttgttta ttgcagctta taatggttac aaataaagca atagcatcac
    10141 aaatttcaca aataaagcat ttttttcact gcattctagt tgtggtttgt ccaaactcat
    10201 caatgtatct tatcatgtct gtataccgtc gacctctagc tagagcttgg cgtaatcatg
    10261 gtcatagctg tttcctgtgt gaaattgtta tccgctcaca attccacaca acatacgagc
    10321 cggaagcata aagtgtaaag cctggggtgc ctaatgagtg agctaactca cattaattgc
    10381 gttgcgctca ctgcccgctt tccagtcggg aaacctgtcg tgccagctgc attaatgaat
    10441 cggccaacgc gcggggagag gcggtttgcg tattgggcgc tcttccgctt cctcgctcac
    10501 tgactcgctg cgctcggtcg ttcggctgcg gcgagcggta tcagctcact caaaggcggt
    10561 aatacggtta tccacagaat caggggataa cgcaggaaag aacatgtgag caaaaggcca
    10621 gcaaaaggcc aggaaccgta aaaaggccgc gttgctggcg tttttccata ggctccgccc
    10681 ccctgacgag catcacaaaa atcgacgctc aagtcagagg tggcgaaacc cgacaggact
    10741 ataaagatac caggcgtttc cccctggaag ctccctcgtg cgctctcctg ttccgaccct
    10801 gccgcttacc ggatacctgt ccgcctttct cccttcggga agcgtggcgc tttctcatag
    10861 ctcacgctgt aggtatctca gttcggtgta ggtcgttcgc tccaagctgg gctgtgtgca
    10921 cgaacccccc gttcagcccg accgctgcgc cttatccggt aactatcgtc ttgagtccaa
    10981 cccggtaaga cacgacttat cgccactggc agcagccact ggtaacagga ttagcagagc
    11041 gaggtatgta ggcggtgcta cagagttctt gaagtggtgg cctaactacg gctacactag
    11101 aagaacagta tttggtatct gcgctctgct gaagccagtt accttcggaa aaagagttgg
    11161 tagctcttga tccggcaaac aaaccaccgc tggtagcggt ggtttttttg tttgcaagca
    11221 gcagattacg cgcagaaaaa aaggatctca agaagatcct ttgatctttt ctacggggtc
    11281 tgacgctcag tggaacgaaa actcacgtta agggattttg gtcatgagat tatcaaaaag
    11341 gatcttcacc tagatccttt taaattaaaa atgaagtttt aaatcaatct aaagtatata
    11401 tgagtaaact tggtctgaca gttaccaatg cttaatcagt gaggcaccta tctcagcgat
    11461 ctgtctattt cgttcatcca tagttgcctg actccccgtc gtgtagataa ctacgatacg
    11521 ggagggctta ccatctggcc ccagtgctgc aatgataccg cgagacccac gctcaccggc
    11581 tccagattta tcagcaataa accagccagc cggaagggcc gagcgcagaa gtggtcctgc
    11641 aactttatcc gcctccatcc agtctattaa ttgttgccgg gaagctagag taagtagttc
    11701 gccagttaat agtttgcgca acgttgttgc cattgctaca ggcatcgtgg tgtcacgctc
    11761 gtcgtttggt atggcttcat tcagctccgg ttcccaacga tcaaggcgag ttacatgatc
    11821 ccccatgttg tgcaaaaaag cggttagctc cttcggtcct ccgatcgttg tcagaagtaa
    11881 gttggccgca gtgttatcac tcatggttat ggcagcactg cataattctc ttactgtcat
    11941 gccatccgta agatgctttt ctgtgactgg tgagtactca accaagtcat tctgagaata
    12001 gtgtatgcgg cgaccgagtt gctcttgccc ggcgtcaata cgggataata ccgcgccaca
    12061 tagcagaact ttaaaagtgc tcatcattgg aaaacgttct tcggggcgaa aactctcaag
    12121 gatcttaccg ctgttgagat ccagttcgat gtaacccact cgtgcaccca actgatcttc
    12181 agcatctttt actttcacca gcgtttctgg gtgagcaaaa acaggaaggc aaaatgccgc
    12241 aaaaaaggga ataagggcga cacggaaatg ttgaatactc atactcttcc tttttcaata
    12301 ttattgaagc atttatcagg gttattgtct catgagcgga tacatatttg aatgtattta
    12361 gaaaaataaa caaatagggg ttccgcgcac atttccccga aaagtgccac ctgacgtcga
    12421 cggatcggga gatctcccga tcccctatgg tgcactctca gtacaatctg ctctgatgcc
    12481 gcatagttaa gccagtatct gctccctgct tgtgtgttgg aggtcgctga gtagtgcgcg
    12541 agcaaaattt aagctacaac aaggcaaggc ttgaccgaca attgcatgaa gaatctgctt
    12601 agggttaggc gttttgcgct gcttcgcgat gtacgggcca gatatacgcg ttgacattga
    12661 ttattgacta gttattaata gtaatcaatt acggggtcat tagttcatag cccatatatg
    12721 gagttccgcg ttacataact tacggtaaat ggcccgcctg gctgaccgcc caacgacccc
    12781 cgcccattga cgtcaataat gacgtatgtt cccatagtaa cgccaatagg gactttccat
    12841 tgacgtcaat gggtggagta tttacggtaa actgcccact tggcagtaca tcaagtgtat
    12901 catatgccaa gtacgccccc tattgacgtc aatgacggta aatggcccgc ctggcattat
    12961 gcccagtaca tgaccttatg ggactttcct acttggcagt acatctacgt att
//
