LOCUS       sequence-90021-e       11232 bp    DNA     circular SYN 15-DEC-2025
DEFINITION  Lentiviral Vector for SpCas9 Expression without sgRNA, Puromycin 
            resistance, EFS Promoter driven.
ACCESSION   .
VERSION     .
KEYWORDS    .
SOURCE      synthetic DNA construct
  ORGANISM  synthetic DNA construct
REFERENCE   1  (bases 1 to 11232)
  AUTHORS   Heckl D, Kowalczyk MS, Yudovich D, Belizaire R, Puram RV, McConkey 
            ME, Thielke A, Aster JC, Regev A, Ebert BL
  TITLE     Generation of mouse models of myeloid malignancy with combinatorial 
            genetic lesions using CRISPR-Cas9 genome editing.
  JOURNAL   Nat Biotechnol. 2014 Jun 22. doi: 10.1038/nbt.2951.
  PUBMED    24952903
REFERENCE   2  (bases 1 to 11232)
  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"; journalName: "Nat 
            Biotechnol. 2014 Jun 22. doi: 10.1038/nbt.2951."
COMMENT     Sequence Label: pLKO5d.EFS.SpCas9.P2A.PAC
FEATURES             Location/Qualifiers
     source          1..11232
                     /mol_type="other DNA"
                     /organism="synthetic DNA construct"
     misc_feature    41..158
                     /label=cPPT/CTS
                     /note="central polypurine tract and central termination 
                     sequence of HIV-1"
     promoter        227..438
                     /label=EF-1-alpha core promoter
                     /note="core promoter for human elongation factor 
                     EF-1-alpha"
     regulatory      458..467
                     /label=Kozak sequence
                     /note="vertebrate consensus sequence for strong initiation 
                     of translation (Kozak, 1987)"
                     /regulatory_class="other"
     CDS             467..490
                     /codon_start=1
                     /product="FLAG(R) epitope tag, followed by an enterokinase 
                     cleavage site"
                     /label=FLAG
                     /translation="DYKDDDDK"
     CDS             497..517
                     /codon_start=1
                     /product="nuclear localization signal of SV40 (simian virus
                     40) large T antigen"
                     /label=SV40 NLS
                     /translation="PKKKRKV"
     CDS             542..4642
                     /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="DKKYSIGLDIGTNSVGWAVITDEYKVPSKKFKVLGNTDRHSIKKN
                     LIGALLFDSGETAEATRLKRTARRRYTRRKNRICYLQEIFSNEMAKVDDSFFHRLEESF
                     LVEEDKKHERHPIFGNIVDEVAYHEKYPTIYHLRKKLVDSTDKADLRLIYLALAHMIKF
                     RGHFLIEGDLNPDNSDVDKLFIQLVQTYNQLFEENPINASGVDAKAILSARLSKSRRLE
                     NLIAQLPGEKKNGLFGNLIALSLGLTPNFKSNFDLAEDAKLQLSKDTYDDDLDNLLAQI
                     GDQYADLFLAAKNLSDAILLSDILRVNTEITKAPLSASMIKRYDEHHQDLTLLKALVRQ
                     QLPEKYKEIFFDQSKNGYAGYIDGGASQEEFYKFIKPILEKMDGTEELLVKLNREDLLR
                     KQRTFDNGSIPHQIHLGELHAILRRQEDFYPFLKDNREKIEKILTFRIPYYVGPLARGN
                     SRFAWMTRKSEETITPWNFEEVVDKGASAQSFIERMTNFDKNLPNEKVLPKHSLLYEYF
                     TVYNELTKVKYVTEGMRKPAFLSGEQKKAIVDLLFKTNRKVTVKQLKEDYFKKIECFDS
                     VEISGVEDRFNASLGTYHDLLKIIKDKDFLDNEENEDILEDIVLTLTLFEDREMIEERL
                     KTYAHLFDDKVMKQLKRRRYTGWGRLSRKLINGIRDKQSGKTILDFLKSDGFANRNFMQ
                     LIHDDSLTFKEDIQKAQVSGQGDSLHEHIANLAGSPAIKKGILQTVKVVDELVKVMGRH
                     KPENIVIEMARENQTTQKGQKNSRERMKRIEEGIKELGSQILKEHPVENTQLQNEKLYL
                     YYLQNGRDMYVDQELDINRLSDYDVDHIVPQSFLKDDSIDNKVLTRSDKNRGKSDNVPS
                     EEVVKKMKNYWRQLLNAKLITQRKFDNLTKAERGGLSELDKAGFIKRQLVETRQITKHV
                     AQILDSRMNTKYDENDKLIREVKVITLKSKLVSDFRKDFQFYKVREINNYHHAHDAYLN
                     AVVGTALIKKYPKLESEFVYGDYKVYDVRKMIAKSEQEIGKATAKYFFYSNIMNFFKTE
                     ITLANGEIRKRPLIETNGETGEIVWDKGRDFATVRKVLSMPQVNIVKKTEVQTGGFSKE
                     SILPKRNSDKLIARKKDWDPKKYGGFDSPTVAYSVLVVAKVEKGKSKKLKSVKELLGIT
                     IMERSSFEKNPIDFLEAKGYKEVKKDLIIKLPKYSLFELENGRKRMLASAGELQKGNEL
                     ALPSKYVNFLYLASHYEKLKGSPEDNEQKQLFVEQHKHYLDEIIEQISEFSKRVILADA
                     NLDKVLSAYNKHRDKPIREQAENIIHLFTLTNLGAPAAFKYFDTTIDRKRYTSTKEVLD
                     ATLIHQSITGLYETRIDLSQLGGD"
     CDS             542..4642
                     /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="DKKYSIGLDIGTNSVGWAVITDEYKVPSKKFKVLGNTDRHSIKKN
                     LIGALLFDSGETAEATRLKRTARRRYTRRKNRICYLQEIFSNEMAKVDDSFFHRLEESF
                     LVEEDKKHERHPIFGNIVDEVAYHEKYPTIYHLRKKLVDSTDKADLRLIYLALAHMIKF
                     RGHFLIEGDLNPDNSDVDKLFIQLVQTYNQLFEENPINASGVDAKAILSARLSKSRRLE
                     NLIAQLPGEKKNGLFGNLIALSLGLTPNFKSNFDLAEDAKLQLSKDTYDDDLDNLLAQI
                     GDQYADLFLAAKNLSDAILLSDILRVNTEITKAPLSASMIKRYDEHHQDLTLLKALVRQ
                     QLPEKYKEIFFDQSKNGYAGYIDGGASQEEFYKFIKPILEKMDGTEELLVKLNREDLLR
                     KQRTFDNGSIPHQIHLGELHAILRRQEDFYPFLKDNREKIEKILTFRIPYYVGPLARGN
                     SRFAWMTRKSEETITPWNFEEVVDKGASAQSFIERMTNFDKNLPNEKVLPKHSLLYEYF
                     TVYNELTKVKYVTEGMRKPAFLSGEQKKAIVDLLFKTNRKVTVKQLKEDYFKKIECFDS
                     VEISGVEDRFNASLGTYHDLLKIIKDKDFLDNEENEDILEDIVLTLTLFEDREMIEERL
                     KTYAHLFDDKVMKQLKRRRYTGWGRLSRKLINGIRDKQSGKTILDFLKSDGFANRNFMQ
                     LIHDDSLTFKEDIQKAQVSGQGDSLHEHIANLAGSPAIKKGILQTVKVVDELVKVMGRH
                     KPENIVIEMARENQTTQKGQKNSRERMKRIEEGIKELGSQILKEHPVENTQLQNEKLYL
                     YYLQNGRDMYVDQELDINRLSDYDVDHIVPQSFLKDDSIDNKVLTRSDKNRGKSDNVPS
                     EEVVKKMKNYWRQLLNAKLITQRKFDNLTKAERGGLSELDKAGFIKRQLVETRQITKHV
                     AQILDSRMNTKYDENDKLIREVKVITLKSKLVSDFRKDFQFYKVREINNYHHAHDAYLN
                     AVVGTALIKKYPKLESEFVYGDYKVYDVRKMIAKSEQEIGKATAKYFFYSNIMNFFKTE
                     ITLANGEIRKRPLIETNGETGEIVWDKGRDFATVRKVLSMPQVNIVKKTEVQTGGFSKE
                     SILPKRNSDKLIARKKDWDPKKYGGFDSPTVAYSVLVVAKVEKGKSKKLKSVKELLGIT
                     IMERSSFEKNPIDFLEAKGYKEVKKDLIIKLPKYSLFELENGRKRMLASAGELQKGNEL
                     ALPSKYVNFLYLASHYEKLKGSPEDNEQKQLFVEQHKHYLDEIIEQISEFSKRVILADA
                     NLDKVLSAYNKHRDKPIREQAENIIHLFTLTNLGAPAAFKYFDTTIDRKRYTSTKEVLD
                     ATLIHQSITGLYETRIDLSQLGGD"
     CDS             4643..4690
                     /codon_start=1
                     /product="bipartite nuclear localization signal from 
                     nucleoplasmin"
                     /label=nucleoplasmin NLS
                     /translation="KRPAATKKAGQAKKKK"
     CDS             4706..4762
                     /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             4763..5362
                     /codon_start=1
                     /gene="pac from Streptomyces alboniger"
                     /product="puromycin N-acetyltransferase"
                     /label=PuroR
                     /note="confers resistance to puromycin"
                     /translation="MTEYKPTVRLATRDDVPRAVRTLAAAFADYPATRHTVDPDRHIER
                     VTELQELFLTRVGLDIGKVWVADDGAAVAVWTTPESVEAGAVFAEIGPRMAELSGSRLA
                     AQQQMEGLLAPHRPKEPAWFLATVGVSPDHQGKGLGSAVVLPGVEAAERAGVPAFLETS
                     APRNLPFYERLGFTVTADVEVPEGPRTWCMTRKPGA"
     primer_bind     complement(4763..4782)
                     /label=Puro-R
                     /note="Puromycin resistance gene, reverse primer. Also 
                     called puro-variant-R"
     primer_bind     5259..5279
                     /label=Puro-F
                     /note="Puromycin resistance gene, forward primer"
     misc_feature    5378..5966
                     /label=WPRE
                     /note="woodchuck hepatitis virus posttranscriptional 
                     regulatory element"
     primer_bind     complement(5431..5451)
                     /label=WPRE-R
                     /note="WPRE, reverse primer"
     CDS             complement(5849..5860)
                     /codon_start=1
                     /product="Factor Xa recognition and cleavage site"
                     /label=Factor Xa site
                     /translation="IEGR"
     LTR             6038..6271
                     /label=3' LTR (Delta-U3)
                     /note="self-inactivating 3' long terminal repeat (LTR) from
                     HIV-1"
     polyA_signal    6349..6470
                     /label=SV40 poly(A) signal
                     /note="SV40 polyadenylation signal"
     primer_bind     complement(6386..6405)
                     /label=SV40pA-R
                     /note="SV40 polyA, reverse primer"
     primer_bind     6440..6459
                     /label=EBV-rev
                     /note="SV40 polyA terminator, reverse primer"
     rep_origin      6510..6645
                     /label=SV40 ori
                     /note="SV40 origin of replication"
     primer_bind     complement(6665..6684)
                     /label=T7
                     /note="T7 promoter, forward primer"
     promoter        complement(6666..6684)
                     /label=T7 promoter
                     /note="promoter for bacteriophage T7 RNA polymerase"
     primer_bind     complement(6694..6711)
                     /label=M13 Forward
                     /note="In lacZ gene. Also called M13-F20 or M13 (-21) 
                     Forward"
     primer_bind     complement(6694..6710)
                     /label=M13 fwd
                     /note="common sequencing primer, one of multiple similar 
                     variants"
     primer_bind     complement(6703..6725)
                     /label=M13/pUC Forward
                     /note="In lacZ gene"
     rep_origin      6852..7307
                     /direction=RIGHT
                     /label=f1 ori
                     /note="f1 bacteriophage origin of replication; arrow 
                     indicates direction of (+) strand synthesis"
     primer_bind     complement(6939..6958)
                     /label=F1ori-R
                     /note="F1 origin, reverse primer"
     primer_bind     7149..7170
                     /label=F1ori-F
                     /note="F1 origin, forward primer"
     promoter        7333..7437
                     /gene="bla"
                     /label=AmpR promoter
     CDS             join(7438..7506,7507..8298)
                     /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     complement(7656..7675)
                     /label=Amp-R
                     /note="Ampicillin resistance gene, reverse primer"
     rep_origin      8469..9057
                     /direction=RIGHT
                     /label=ori
                     /note="high-copy-number ColE1/pMB1/pBR322/pUC origin of 
                     replication"
     primer_bind     8958..8977
                     /label=pBR322ori-F
                     /note="pBR322 origin, forward primer"
     primer_bind     9211..9228
                     /label=L4440
                     /note="L4440 vector, forward primer"
     protein_bind    9345..9366
                     /label=CAP binding site
                     /bound_moiety="E. coli catabolite activator protein"
                     /note="CAP binding activates transcription in the presence 
                     of cAMP."
     promoter        join(9381..9386,9387..9404,9405..9411)
                     /label=lac promoter
                     /note="promoter for the E. coli lac operon"
     protein_bind    9419..9435
                     /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)."
     primer_bind     9424..9446
                     /label=M13/pUC Reverse
                     /note="In lacZ gene"
     primer_bind     9443..9459
                     /label=M13 rev
                     /note="common sequencing primer, one of multiple similar 
                     variants"
     primer_bind     9443..9459
                     /label=M13 Reverse
                     /note="In lacZ gene. Also called M13-rev"
     primer_bind     9478..9498
                     /label=T3
                     /note="T3 promoter, forward primer"
     promoter        9480..9498
                     /label=T3 promoter
                     /note="promoter for bacteriophage T3 RNA polymerase"
     promoter        9526..9752
                     /label=RSV promoter
                     /note="Rous sarcoma virus enhancer/promoter"
     LTR             9753..9933
                     /label=5' LTR (truncated)
                     /note="truncated 5' long terminal repeat (LTR) from HIV-1"
     misc_feature    9980..10105
                     /label=HIV-1 Psi
                     /note="packaging signal of human immunodeficiency virus 
                     type 1"
     misc_feature    10598..10831
                     /label=RRE
                     /note="The Rev response element (RRE) of HIV-1 allows for 
                     Rev-dependent mRNA export from the nucleus to the 
                     cytoplasm."
     CDS             11016..11060
                     /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"
ORIGIN
        1 aattctagat cttgagacaa atggcagtat tcatccacaa ttttaaaaga aaagggggga
       61 ttggggggta cagtgcaggg gaaagaatag tagacataat agcaacagac atacaaacta
      121 aagaattaca aaaacaaatt acaaaaattc aaaattttcg ggtttattac agggacagca
      181 gagatccact ttggcgccgg ctcgagtggc tccggtgccc gtcagtgggc agagcgcaca
      241 tcgcccacag tccccgagaa gttgggggga ggggtcggca attgaaccgg tgcctagaga
      301 aggtggcgcg gggtaaactg ggaaagtgat gtcgtgtact ggctccgcct ttttcccgag
      361 ggtgggggag aaccgtatat aagtgcagta gtcgccgtga acgttctttt tcgcaacggg
      421 tttgccgcca gaacacaggt gtcgtgacgc gggatccgcc accatggatt acaaagacga
      481 tgacgataag atggccccaa agaagaagcg gaaggtcggt atccacggag tcccagcagc
      541 cgacaagaag tacagcatcg gcctggacat cggcaccaac tctgtgggct gggccgtgat
      601 caccgacgag tacaaggtgc ccagcaagaa attcaaggtg ctgggcaaca ccgaccggca
      661 cagcatcaag aagaacctga tcggagccct gctgttcgac agcggcgaaa cagccgaggc
      721 cacccggctg aagagaaccg ccagaagaag atacaccaga cggaagaacc ggatctgcta
      781 tctgcaagag atcttcagca acgagatggc caaggtggac gacagcttct tccacagact
      841 ggaagagtcc ttcctggtgg aagaggataa gaagcacgag cggcacccca tcttcggcaa
      901 catcgtggac gaggtggcct accacgagaa gtaccccacc atctaccacc tgagaaagaa
      961 actggtggac agcaccgaca aggccgacct gcggctgatc tatctggccc tggcccacat
     1021 gatcaagttc cggggccact tcctgatcga gggcgacctg aaccccgaca acagcgacgt
     1081 ggacaagctg ttcatccagc tggtgcagac ctacaaccag ctgttcgagg aaaaccccat
     1141 caacgccagc ggcgtggacg ccaaggccat cctgtctgcc agactgagca agagcagacg
     1201 gctggaaaat ctgatcgccc agctgcccgg cgagaagaag aatggcctgt tcggcaacct
     1261 gattgccctg agcctgggcc tgacccccaa cttcaagagc aacttcgacc tggccgagga
     1321 tgccaaactg cagctgagca aggacaccta cgacgacgac ctggacaacc tgctggccca
     1381 gatcggcgac cagtacgccg acctgtttct ggccgccaag aacctgtccg acgccatcct
     1441 gctgagcgac atcctgagag tgaacaccga gatcaccaag gcccccctga gcgcctctat
     1501 gatcaagaga tacgacgagc accaccagga cctgaccctg ctgaaagctc tcgtgcggca
     1561 gcagctgcct gagaagtaca aagagatttt cttcgaccag agcaagaacg gctacgccgg
     1621 ctacattgac ggcggagcca gccaggaaga gttctacaag ttcatcaagc ccatcctgga
     1681 aaagatggac ggcaccgagg aactgctcgt gaagctgaac agagaggacc tgctgcggaa
     1741 gcagcggacc ttcgacaacg gcagcatccc ccaccagatc cacctgggag agctgcacgc
     1801 cattctgcgg cggcaggaag atttttaccc attcctgaag gacaaccggg aaaagatcga
     1861 gaagatcctg accttccgca tcccctacta cgtgggccct ctggccaggg gaaacagcag
     1921 attcgcctgg atgaccagaa agagcgagga aaccatcacc ccctggaact tcgaggaagt
     1981 ggtggacaag ggcgcttccg cccagagctt catcgagcgg atgaccaact tcgataagaa
     2041 cctgcccaac gagaaggtgc tgcccaagca cagcctgctg tacgagtact tcaccgtgta
     2101 taacgagctg accaaagtga aatacgtgac cgagggaatg agaaagcccg ccttcctgag
     2161 cggcgagcag aaaaaggcca tcgtggacct gctgttcaag accaaccgga aagtgaccgt
     2221 gaagcagctg aaagaggact acttcaagaa aatcgagtgc ttcgactccg tggaaatctc
     2281 cggcgtggaa gatcggttca acgcctccct gggcacatac cacgatctgc tgaaaattat
     2341 caaggacaag gacttcctgg acaatgagga aaacgaggac attctggaag atatcgtgct
     2401 gaccctgaca ctgtttgagg acagagagat gatcgaggaa cggctgaaaa cctatgccca
     2461 cctgttcgac gacaaagtga tgaagcagct gaagcggcgg agatacaccg gctggggcag
     2521 gctgagccgg aagctgatca acggcatccg ggacaagcag tccggcaaga caatcctgga
     2581 tttcctgaag tccgacggct tcgccaacag aaacttcatg cagctgatcc acgacgacag
     2641 cctgaccttt aaagaggaca tccagaaagc ccaggtgtcc ggccagggcg atagcctgca
     2701 cgagcacatt gccaatctgg ccggcagccc cgccattaag aagggcatcc tgcagacagt
     2761 gaaggtggtg gacgagctcg tgaaagtgat gggccggcac aagcccgaga acatcgtgat
     2821 cgaaatggcc agagagaacc agaccaccca gaagggacag aagaacagcc gcgagagaat
     2881 gaagcggatc gaagagggca tcaaagagct gggcagccag atcctgaaag aacaccccgt
     2941 ggaaaacacc cagctgcaga acgagaagct gtacctgtac tacctgcaga atgggcggga
     3001 tatgtacgtg gaccaggaac tggacatcaa ccggctgtcc gactacgatg tggaccatat
     3061 cgtgcctcag agctttctga aggacgactc catcgacaac aaggtgctga ccagaagcga
     3121 caagaaccgg ggcaagagcg acaacgtgcc ctccgaagag gtcgtgaaga agatgaagaa
     3181 ctactggcgg cagctgctga acgccaagct gattacccag agaaagttcg acaatctgac
     3241 caaggccgag agaggcggcc tgagcgaact ggataaggcc ggcttcatca agagacagct
     3301 ggtggaaacc cggcagatca caaagcacgt ggcacagatc ctggactccc ggatgaacac
     3361 taagtacgac gagaatgaca agctgatccg ggaagtgaaa gtgatcaccc tgaagtccaa
     3421 gctggtgtcc gatttccgga aggatttcca gttttacaaa gtgcgcgaga tcaacaacta
     3481 ccaccacgcc cacgacgcct acctgaacgc cgtcgtggga accgccctga tcaaaaagta
     3541 ccctaagctg gaaagcgagt tcgtgtacgg cgactacaag gtgtacgacg tgcggaagat
     3601 gatcgccaag agcgagcagg aaatcggcaa ggctaccgcc aagtacttct tctacagcaa
     3661 catcatgaac tttttcaaga ccgagattac cctggccaac ggcgagatcc ggaagcggcc
     3721 tctgatcgag acaaacggcg aaaccgggga gatcgtgtgg gataagggcc gggattttgc
     3781 caccgtgcgg aaagtgctga gcatgcccca agtgaatatc gtgaaaaaga ccgaggtgca
     3841 gacaggcggc ttcagcaaag agtctatcct gcccaagagg aacagcgata agctgatcgc
     3901 cagaaagaag gactgggacc ctaagaagta cggcggcttc gacagcccca ccgtggccta
     3961 ttctgtgctg gtggtggcca aagtggaaaa gggcaagtcc aagaaactga agagtgtgaa
     4021 agagctgctg gggatcacca tcatggaaag aagcagcttc gagaagaatc ccatcgactt
     4081 tctggaagcc aagggctaca aagaagtgaa aaaggacctg atcatcaagc tgcctaagta
     4141 ctccctgttc gagctggaaa acggccggaa gagaatgctg gcctctgccg gcgaactgca
     4201 gaagggaaac gaactggccc tgccctccaa atatgtgaac ttcctgtacc tggccagcca
     4261 ctatgagaag ctgaagggct cccccgagga taatgagcag aaacagctgt ttgtggaaca
     4321 gcacaagcac tacctggacg agatcatcga gcagatcagc gagttctcca agagagtgat
     4381 cctggccgac gctaatctgg acaaagtgct gtccgcctac aacaagcacc gggataagcc
     4441 catcagagag caggccgaga atatcatcca cctgtttacc ctgaccaatc tgggagcccc
     4501 tgccgccttc aagtactttg acaccaccat cgaccggaag aggtacacca gcaccaaaga
     4561 ggtgctggac gccaccctga tccaccagag catcaccggc ctgtacgaga cacggatcga
     4621 cctgtctcag ctgggaggcg acaagcgtcc tgctgctact aagaaagctg gtcaagctaa
     4681 gaaaaagaaa gctagcggca gcggcgccac caacttcagc ctgctgaagc aggccggcga
     4741 cgtggaggag aaccccggcc ccatgaccga gtacaagccc acggtgcgcc tcgccacccg
     4801 cgacgacgtc cccagggccg tacgcaccct cgccgccgcg ttcgccgact accccgccac
     4861 gcgccacacc gtcgatccgg accgccacat cgagcgggtc accgagctgc aagaactctt
     4921 cctcacgcgc gtcgggctcg acatcggcaa ggtgtgggtc gcggacgacg gcgccgcggt
     4981 ggcggtctgg accacgccgg agagcgtcga agcgggggcg gtgttcgccg agatcggccc
     5041 gcgcatggcc gagttgagcg gttcccggct ggccgcgcag caacagatgg aaggcctcct
     5101 ggcgccgcac cggcccaagg agcccgcgtg gttcctggcc accgtcggcg tctcgcccga
     5161 ccaccagggc aagggtctgg gcagcgccgt cgtgctcccc ggagtggagg cggccgagcg
     5221 cgccggggtg cccgccttcc tggagacctc cgcgccccgc aacctcccct tctacgagcg
     5281 gctcggcttc accgtcaccg ccgacgtcga ggtgcccgaa ggaccgcgca cctggtgcat
     5341 gacccgcaag cccggtgcct gaacgcgtta agtcgacaat caacctctgg attacaaaat
     5401 ttgtgaaaga ttgactggta ttcttaacta tgttgctcct tttacgctat gtggatacgc
     5461 tgctttaatg cctttgtatc atgctattgc ttcccgtatg gctttcattt tctcctcctt
     5521 gtataaatcc tggttgctgt ctctttatga ggagttgtgg cccgttgtca ggcaacgtgg
     5581 cgtggtgtgc actgtgtttg ctgacgcaac ccccactggt tggggcattg ccaccacctg
     5641 tcagctcctt tccgggactt tcgctttccc cctccctatt gccacggcgg aactcatcgc
     5701 cgcctgcctt gcccgctgct ggacaggggc tcggctgttg ggcactgaca attccgtggt
     5761 gttgtcgggg aaatcatcgt cctttccttg gctgctcgcc tgtgttgcca cctggattct
     5821 gcgcgggacg tccttctgct acgtcccttc ggccctcaat ccagcggacc ttccttcccg
     5881 cggcctgctg ccggctctgc ggcctcttcc gcgtcttcgc cttcgccctc agacgagtcg
     5941 gatctccctt tgggccgcct ccccgcgtcg actttaagac caatgactta caaggcagct
     6001 gtagatctta gccacttttt aaaagaaaag gggggactgg aagggctaat tcactcccaa
     6061 cgaagacaag atctgctttt tgcttgtact gggtctctct ggttagacca gatctgagcc
     6121 tgggagctct ctggctaact agggaaccca ctgcttaagc ctcaataaag cttgccttga
     6181 gtgcttcaag tagtgtgtgc ccgtctgttg tgtgactctg gtaactagag atccctcaga
     6241 cccttttagt cagtgtggaa aatctctagc agtacgtata gtagttcatg tcatcttatt
     6301 attcagtatt tataacttgc aaagaaatga atatcagaga gtgagaggaa cttgtttatt
     6361 gcagcttata atggttacaa ataaagcaat agcatcacaa atttcacaaa taaagcattt
     6421 ttttcactgc attctagttg tggtttgtcc aaactcatca atgtatctta tcatgtctgg
     6481 ctctagctat cccgccccta actccgccca tcccgcccct aactccgccc agttccgccc
     6541 attctccgcc ccatggctga ctaatttttt ttatttatgc agaggccgag gccgcctcgg
     6601 cctctgagct attccagaag tagtgaggag gcttttttgg aggcctaggg acgtacccaa
     6661 ttcgccctat agtgagtcgt attacgcgcg ctcactggcc gtcgttttac aacgtcgtga
     6721 ctgggaaaac cctggcgtta cccaacttaa tcgccttgca gcacatcccc ctttcgccag
     6781 ctggcgtaat agcgaagagg cccgcaccga tcgcccttcc caacagttgc gcagcctgaa
     6841 tggcgaatgg gacgcgccct gtagcggcgc attaagcgcg gcgggtgtgg tggttacgcg
     6901 cagcgtgacc gctacacttg ccagcgccct agcgcccgct cctttcgctt tcttcccttc
     6961 ctttctcgcc acgttcgccg gctttccccg tcaagctcta aatcgggggc tccctttagg
     7021 gttccgattt agtgctttac ggcacctcga ccccaaaaaa cttgattagg gtgatggttc
     7081 acgtagtggg ccatcgccct gatagacggt ttttcgccct ttgacgttgg agtccacgtt
     7141 ctttaatagt ggactcttgt tccaaactgg aacaacactc aaccctatct cggtctattc
     7201 ttttgattta taagggattt tgccgatttc ggcctattgg ttaaaaaatg agctgattta
     7261 acaaaaattt aacgcgaatt ttaacaaaat attaacgctt acaatttagg tggcactttt
     7321 cggggaaatg tgcgcggaac ccctatttgt ttatttttct aaatacattc aaatatgtat
     7381 ccgctcatga gacaataacc ctgataaatg cttcaataat attgaaaaag gaagagtatg
     7441 agtattcaac atttccgtgt cgcccttatt cccttttttg cggcattttg ccttcctgtt
     7501 tttgctcacc cagaaacgct ggtgaaagta aaagatgctg aagatcagtt gggtgcacga
     7561 gtgggttaca tcgaactgga tctcaacagc ggtaagatcc ttgagagttt tcgccccgaa
     7621 gaacgttttc caatgatgag cacttttaaa gttctgctat gtggcgcggt attatcccgt
     7681 attgacgccg ggcaagagca actcggtcgc cgcatacact attctcagaa tgacttggtt
     7741 gagtactcac cagtcacaga aaagcatctt acggatggca tgacagtaag agaattatgc
     7801 agtgctgcca taaccatgag tgataacact gcggccaact tacttctgac aacgatcgga
     7861 ggaccgaagg agctaaccgc ttttttgcac aacatggggg atcatgtaac tcgccttgat
     7921 cgttgggaac cggagctgaa tgaagccata ccaaacgacg agcgtgacac cacgatgcct
     7981 gtagcaatgg caacaacgtt gcgcaaacta ttaactggcg aactacttac tctagcttcc
     8041 cggcaacaat taatagactg gatggaggcg gataaagttg caggaccact tctgcgctcg
     8101 gcccttccgg ctggctggtt tattgctgat aaatctggag ccggtgagcg tgggtctcgc
     8161 ggtatcattg cagcactggg gccagatggt aagccctccc gtatcgtagt tatctacacg
     8221 acggggagtc aggcaactat ggatgaacga aatagacaga tcgctgagat aggtgcctca
     8281 ctgattaagc attggtaact gtcagaccaa gtttactcat atatacttta gattgattta
     8341 aaacttcatt tttaatttaa aaggatctag gtgaagatcc tttttgataa tctcatgacc
     8401 aaaatccctt aacgtgagtt ttcgttccac tgagcgtcag accccgtaga aaagatcaaa
     8461 ggatcttctt gagatccttt ttttctgcgc gtaatctgct gcttgcaaac aaaaaaacca
     8521 ccgctaccag cggtggtttg tttgccggat caagagctac caactctttt tccgaaggta
     8581 actggcttca gcagagcgca gataccaaat actgttcttc tagtgtagcc gtagttaggc
     8641 caccacttca agaactctgt agcaccgcct acatacctcg ctctgctaat cctgttacca
     8701 gtggctgctg ccagtggcga taagtcgtgt cttaccgggt tggactcaag acgatagtta
     8761 ccggataagg cgcagcggtc gggctgaacg gggggttcgt gcacacagcc cagcttggag
     8821 cgaacgacct acaccgaact gagataccta cagcgtgagc tatgagaaag cgccacgctt
     8881 cccgaaggga gaaaggcgga caggtatccg gtaagcggca gggtcggaac aggagagcgc
     8941 acgagggagc ttccaggggg aaacgcctgg tatctttata gtcctgtcgg gtttcgccac
     9001 ctctgacttg agcgtcgatt tttgtgatgc tcgtcagggg ggcggagcct atggaaaaac
     9061 gccagcaacg cggccttttt acggttcctg gccttttgct ggccttttgc tcacatgttc
     9121 tttcctgcgt tatcccctga ttctgtggat aaccgtatta ccgcctttga gtgagctgat
     9181 accgctcgcc gcagccgaac gaccgagcgc agcgagtcag tgagcgagga agcggaagag
     9241 cgcccaatac gcaaaccgcc tctccccgcg cgttggccga ttcattaatg cagctggcac
     9301 gacaggtttc ccgactggaa agcgggcagt gagcgcaacg caattaatgt gagttagctc
     9361 actcattagg caccccaggc tttacacttt atgcttccgg ctcgtatgtt gtgtggaatt
     9421 gtgagcggat aacaatttca cacaggaaac agctatgacc atgattacgc caagcgcgca
     9481 attaaccctc actaaaggga acaaaagctg gagctgcaag cttaatgtag tcttatgcaa
     9541 tactcttgta gtcttgcaac atggtaacga tgagttagca acatgcctta caaggagaga
     9601 aaaagcaccg tgcatgccga ttggtggaag taaggtggta cgatcgtgcc ttattaggaa
     9661 ggcaacagac gggtctgaca tggattggac gaaccactga attgccgcat tgcagagata
     9721 ttgtatttaa gtgcctagct cgatacataa acgggtctct ctggttagac cagatctgag
     9781 cctgggagct ctctggctaa ctagggaacc cactgcttaa gcctcaataa agcttgcctt
     9841 gagtgcttca agtagtgtgt gcccgtctgt tgtgtgactc tggtaactag agatccctca
     9901 gaccctttta gtcagtgtgg aaaatctcta gcagtggcgc ccgaacaggg acttgaaagc
     9961 gaaagggaaa ccagaggagc tctctcgacg caggactcgg cttgctgaag cgcgcacggc
    10021 aagaggcgag gggcggcgac tggtgagtac gccaaaaatt ttgactagcg gaggctagaa
    10081 ggagagagat gggtgcgaga gcgtcagtat taagcggggg agaattagat cgcgatggga
    10141 aaaaattcgg ttaaggccag ggggaaagaa aaaatataaa ttaaaacata tagtatgggc
    10201 aagcagggag ctagaacgat tcgcagttaa tcctggcctg ttagaaacat cagaaggctg
    10261 tagacaaata ctgggacagc tacaaccatc ccttcagaca ggatcagaag aacttagatc
    10321 attatataat acagtagcaa ccctctattg tgtgcatcaa aggatagaga taaaagacac
    10381 caaggaagct ttagacaaga tagaggaaga gcaaaacaaa agtaagacca ccgcacagca
    10441 agcggccgct gatcttcaga cctggaggag gagatatgag ggacaattgg agaagtgaat
    10501 tatataaata taaagtagta aaaattgaac cattaggagt agcacccacc aaggcaaaga
    10561 gaagagtggt gcagagagaa aaaagagcag tgggaatagg agctttgttc cttgggttct
    10621 tgggagcagc aggaagcact atgggcgcag cgtcaatgac gctgacggta caggccagac
    10681 aattattgtc tggtatagtg cagcagcaga acaatttgct gagggctatt gaggcgcaac
    10741 agcatctgtt gcaactcaca gtctggggca tcaagcagct ccaggcaaga atcctggctg
    10801 tggaaagata cctaaaggat caacagctcc tggggatttg gggttgctct ggaaaactca
    10861 tttgcaccac tgctgtgcct tggaatgcta gttggagtaa taaatctctg gaacagattt
    10921 ggaatcacac gacctggatg gagtgggaca gagaaattaa caattacaca agcttaatac
    10981 actccttaat tgaagaatcg caaaaccagc aagaaaagaa tgaacaagaa ttattggaat
    11041 tagataaatg ggcaagtttg tggaattggt ttaacataac aaattggctg tggtatataa
    11101 aattattcat aatgatagta ggaggcttgg taggtttaag aatagttttt gctgtacttt
    11161 ctatagtgaa tagagttagg cagggatatt caccattatc gtttcagacc cacctcccaa
    11221 ccccgagggg ac
//
